Isolated DNA molecules encoding humanized calcitonin gene-related peptide receptor, related non-human transgenic animals and assay methods

ABSTRACT

Disclosed herein are isolated nucleic acid molecules encoding a humanized version of a calcitonin gene-related peptide (CGRP) receptor, which comprises the G-protein coupled receptor calcitonin-receptor-like receptor (CRLR) and the receptor-activity-modifying protein 1 (RAMP1). The humanized CGRP receptors of the present invention attain pharmacological profiles similar to the wild type human receptor via modifications to the respective mammalian RAMP1 nucleotide sequence, specifically at amino acid 74. Also described are related recombinant vectors, recombinant hosts and associated methods for generating such humanized CGRP receptors. Also presented are non-human transgenic animals which express humanized RAMP1. Such animals have been engineered to provide for a CGRP pharmacological profile similar to human CGRP.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit, under 35 U.S.C. §119(e), to U.S.provisional application 60/325,295 filed Sep. 27, 2001.

STATEMENT REGARDING FEDERALLY-SPONSORED R&D

Not Applicable

REFERENCE TO MICROFICHE APPENDIX

Not applicable.

FIELD OF THE INVENTION

The present invention relates in part to isolated nucleic acid molecules(polynucleotides) which encode a humanized version of a calcitoningene-related peptide (CGRP) receptor, which comprises the G-proteincoupled receptor calcitonin-receptor-like receptor (CRLR) and thereceptor-activity-modifying protein 1 (RAMP1). The humanized CGRPreceptors of the present invention attain pharmacological profilessimilar to the wild type human receptor via modifications to therespective mammalian RAMP1 nucleotide sequence. The present inventionalso relates to recombinant vectors and recombinant hosts which containa DNA fragment encoding a humanized version of a CGRP receptor,substantially purified forms of associated humanized version of a CGRPreceptor, recombinant membrane fractions comprising these proteins,associated mutant proteins, and methods associated with identifyingcompounds which specifically modulated human CGRP receptor activityutilizing the humanized version of RAMP1 in various assays. The presentinvention also relates to cells and non-human transgenic animals whereinthe endogenous gene encoding RAMP1 has been engineered to provide for aCGRP receptor pharmacological profile similar to the human CGRPreceptor. Therefore, the transgenic animals of the present inventionwill provide for a phenotype whereby their pharmacological profile inregard to modulators of the CGRP receptor will mimic the human form ofthe receptor, not the form of the CGRP receptor endogenous to thetransgenic animal. The present invention also relates to methods ofscreening for CGRP modulators which comprises utilizing a humanizedversion of the CGRP receptor to selectively identify modulators of humanCGRP. Such CGRP receptor modulators will potentially be useful in thetreatment of various disorders, including but not limited to migraineheadaches, pain indications, menopausal hot flashes, migraineprophylaxis, chronic tension type headache, cluster headache, neurogenicor chronic inflammation, gastrointestinal disorders, type 2 diabetes andcardiovascular disorders.

BACKGROUND OF THE INVENTION

Calcitonin gene-related protein (CGRP) is a 37-amino acid neuropeptidethat is expressed in a variety of cell types in both the central andperipheral nervous systems. In many tissues, CGRP-containing fibers areclosely associated with blood vessels. Among the various physiologicalfunctions reported for CGRP, the most pronounced is vasodilation. CGRPis the most powerful of the vasodilator transmitters and its vasoactiveeffects have been demonstrated in a variety of blood vessels, includingthose in the cerebral, coronary, and mesenteric vasculature.

Mounting evidence suggests that CGRP is involved in the pathophysiologyof migraine headache. Migraine is thought to be associated with dilationof cerebral blood vessels and activation of the trigeminovascularsystem. During the headache phase of a migraine, CGRP levels areelevated in the cranial venous circulation. Successful amelioration ofthe headache results in normalization of CGRP levels, thus implicatingCGRP in the pathophysiology of this disorder. Moreover, intravenousadministration of CGRP to migraineurs induces a delayed migrainousheadache in some patients. These observations suggest that inhibition ofCGRP-mediated vasodilation may have therapeutic utility in the treatmentof migraine headaches, and including but not limited to additionalindication described herein.

Aiyar, et al.(1996, J. Biol. Chem. 271: 11325–11329) disclose the geneencoding the human calcitonin receptor-like receptor (hCRLR).

McLatchie, et al. (1998, Nature 393: 333–339) disclose the gene encodingthe human receptor-activity modifying proteins (hRAMP1).

Luebke, et al. (1996, Proc. Natl. Acad. Sci., USA 93: 3455–3460)disclose the gene encoding the human receptor component protein (hRCP).

The heterodimeric CGRP receptor requires co-expression of calcitoninreceptor-like receptor (CRLR) and an accessory protein called receptoractivity modifying protein-1, or RAMP1. Several small molecule CGRPreceptor antagonists have been shown to exhibit marked speciesselectivity, with >100-fold higher affinities for the human CGRPreceptor than for receptors from other species. CGRP activity ismediated by the G_(s)-coupled G-protein coupled receptor (GPCR), CRLR,which shares 55% homology with the calcitonin receptor. McLatchie et al.(id.) disclose that functional CGRP and adrenomedullin receptors areboth derived from CRLR and that the phenotype is determined byco-expression with a particular RAMP. Co-expression of CRLR with RAMP1results in CGRP receptor pharmacology, while RAMP2 or RAMP3co-expression produces an adrenomedullin receptor. RAMPs are relativelysmall (148–175 amino acids) proteins containing a single predictedmembrane spanning domain, a large extracellular domain, and a shortcytoplasmic domain. The molecular function of RAMPs includescell-surface targeting and may involve direct ligand binding or indirectmodulation of CRLR conformation, or both.

Doods, et al. (2000, Br. J. Pharmacol. 129: 420–423) disclose that aknown small-molecule antagonist of the CGRP receptor demonstrates highaffinity for the human CGRP receptor, with a K_(i) of 14 pM. Ofparticular interest was the observation that this compound exhibited200-fold lower affinity for CGRP receptors from rat, rabbit, dog, andguinea pig, although the affinity for the marmoset receptor was reportedto be similar to that for human. These authors then utilized marmosetfor in vivo studies to evaluate the utility of BIBN4096BS as a potentialanti-migraine agent.

It is desirable to discover new drugs which antagonize the CGRP receptorfor the treatment of various disorders, including but not limited tomigraine, pain, menopausal hot flash, migraine prophylaxis, chronictension type headache, cluster headache, neurogenic or chronicinflammation, gastrointestinal disorders, type 2 diabetes, as well asCGRP agonists which may be useful in the treatment of variouscardiovascular disorders. To this end, it is imperative to develop aconvenient animal model which expresses a CGRP receptor that mimicshuman CGRP pharmacological profiles, thus allowing for in vivo efficacyand receptor occupancy studies for testing of potential modulators ofCGRP receptor activity, especially human CGRP activity. The presentinvention addresses and meets these needs by disclosing a “humanized”version of mammalian RAMP1. Co-expression of such a RAMP1 mutant with amammalian form of CRLR results in a CGRP receptor in which smallmolecule CGRP receptor antagonists display potency similar to that forthe human CGRP receptor. Such a mutant will be useful in both variousscreening assays which are known in the art, such as cell based assays,receptor binding assays and/or radioligand binding assays, as well asthe generation of transgenic animals which provide for this humanizedCGRP receptor activity.

SUMMARY OF THE INVENTION

The present invention relates to an isolated or purified nucleic acidmolecule (polynucleotide) which encodes a humanized version of thereceptor-activity-modifying protein1 (RAMP1).

The present invention further relates to non-human animal cells,non-human transgenic animals, such as founders and littermates,especially transgenic “knock-in” animals, wherein the endogenous geneencoding RAMP1 has been engineered (i.e., “humanized”) to provide for aCGRP receptor pharmacological profile similar to human CGRP receptor. Apreferred transgenic animal for the construction of such a targeted“knock-in” is a mouse.

The present invention relates to isolated or purified mammalian nucleicacid molecules which encode a chimeric, hybrid and/or mutant version ofa mammalian RAMP1 protein, wherein such a derivative RAMP1 proteincomprises the respective mammalian amino acid sequence at least fromabout amino acid 1 to amino acid 65 and from about amino acid 113 toabout amino acid 148, wherein the region corresponding from about aminoacid 66 to amino acid 112 is at least partially derived from the humanRAMP1 coding region.

The present invention further relates to isolated or purified mammaliannucleic acid molecules which encode a chimeric, hybrid and/or mutantversion of a mammalian RAMP1 protein, wherein such a derivative RAMP1protein at least comprises a nucleotide change which results in analteration of amino acid residue 74 to a tryptophan residue, whichresults in a humanized mammalian form of RAMP1, exemplified herein by,but not limited to, the nucleic acid molecules disclosed as SEQ ID NOs1, 3, 5 and 7.

The present invention also relates to fragments or portions of ahumanized RAMP1 nucleotide sequence which encompasses the region whichencodes the “humanizing” amino acid residue, namely the amino acidresidue which corresponds to amino acid 74 of the human RAMP1 proteinand which has been altered to encode a tryptophan residue in therespective mammalian RAMP1 nucleotide sequence, including but notlimited to such fragments generated from SEQ ID NOs 1, 3, 5 and 7 whichencompass the region encoding amino acid residue 74, shown herein to beresponsible for “humanization” of the expressed mammalian RAMP1 protein.

The present invention also relates to recombinant vectors andrecombinant host cells, both prokaryotic and eukaryotic, which have beentransformed or transfected to contain the nucleic acid moleculesdisclosed throughout this specification and which encode a humanizedversion of a CGRP receptor and associated fragment thereof,substantially purified forms of a humanized version of a CGRP receptor,recombinant membrane fractions comprising these proteins (e.g., activeCGRP receptors comprising CRLR and humanized RAMP1 proteins), associatedmutant proteins, and methods associated with identifying compounds whichspecifically modulated human CGRP utilizing the humanized version ofCGRP receptor in various assays.

The present invention also relates to a substantially purified form of ahumanized RAMP1 protein, including but not limited to a substantiallypurified, fully processed (including proteolytic processing,glycosylation and/or phosphorylation), mature humanized RAMP1 proteinobtained from a recombinant host cell.

The present invention further relates to a substantially purifiedmembrane preparation, partially purified membrane preparation, or celllysate which has been obtained from a recombinant host cell transformedor transfected with a DNA expression vector which comprises andappropriately expresses a humanized RAMP1 protein. As noted above, it ispreferred that such membrane preparations comprise both a respectivemammalian CRLR and RAMP1 protein, so as to form an active, humanizedCGRP receptor.

The present invention also relates to biologically active fragmentsand/or mutants of a humanized RAMP1 protein, comprising and/orconsisting of the amino acid sequence as set forth in SEQ ID NOs: 2, 4,6, and/or 8.

The present invention also relates to polyclonal and monoclonalantibodies raised against forms of humanized RAMP1, a biologicallyactive fragment of humanized RAMP1, and/or a CGRP receptor complex whichcomprises a humanized RAMP1.

The present invention also relates to isolated nucleic acid moleculeswhich encode humanized RAMP1 fusion constructs.

It is an object of the present invention to provide an isolated nucleicacid molecule (including but not limited to SEQ ID NOs: 1, 3, 5, and/or7) which encodes a humanized version of RAMP1, or fragments, mutants orderivatives of RAMP1, as set forth in SEQ ID NOs: 2, 4, 6 and 8,respectively. Any such polynucleotide includes but is not necessarilylimited to nucleotide substitutions, deletions, additions,amino-terminal truncations and carboxy-terminal truncations such thatthese mutations encode mRNA which express a protein or protein fragment,which upon co-expression with a mammalian CRLR protein, may exhibitpharmacological properties similar to the human CGRP receptor.

It is an especially preferred object of the present invention to providefor non-human transgenic animals wherein a “humanized” version of RAMP1is co-expressed with endogenous CRLR, or more preferably, a “knock-in”of the humanized transgene (or a portion comprising amino acid residue74) to replace the complementary endogenous sequence is performed.

It is a further object of the present invention to provide the humanizedRAMP1 proteins or protein fragments encoded by the nucleic acidmolecules referred to in the preceding paragraph.

It is another object of the present invention to provide recombinantvectors and recombinant host cells which comprise a nucleic acidsequence encoding a humanized version of RAMP1 or a biologicalequivalent thereof.

It is an object of the present invention to provide a substantiallypurified form of humanized RAMP1 proteins, including but not limited tothose set forth in SEQ ID NOs: 2, 4, 6 and 8.

Is another object of the present invention to provide a substantiallypurified recombinant form of a humanized version RAMP1 protein which hasbeen obtained from a recombinant host cell transformed or transfectedwith a DNA expression vector which comprises and appropriately expressesa complete open reading frame of a mammalian RAMP1 gene, including butin no way limited to DNA expression vectors which comprise nucleic acidmolecules as set forth in SEQ ID NOs: 1, 3 5, and 7, respectively,resulting in a functional, processed form of the respective humanizedRAMP1. As discussed herein, it is preferred that the RAMP1 protein ofthe present invention be co-expressed with a mammalian form of CRLR. Tothis end it is further an object of the present invention to provide forsubstantially purified subcellular membrane preparations, partiallypurified subcellular membrane preparations, or crude lysates fromrecombinant cells which comprise pharmacologically active humanized CGRPreceptor, which comprises CRLR and humanized RAMP1 of the presentinvention. It is also preferred that the recombinant host cell be from aeukaryotic host cell line, such as a mammalian cell line.

It is also an object of the present invention to use cells expressingpharmacologically active humanized CGRP receptor or membranepreparations containing pharmacologically active humanized CGRP receptoror a biological equivalent to screen for modulators, preferablyselective antagonists, of CGRP activity. Any such protein, proteincomplex or membrane associated protein receptor may be useful inscreening and selecting CGRP antagonists for the treatment of variousconditions as mentioned herein.

As used herein, “isolated or purified nucleic acid molecule” means atleast 90%, preferably 95%, more preferably 99%, and even more preferably99.9%, free of other nucleic acids. As used interchangeably with theterms “substantially free from other nucleic acids” or “substantiallypurified” or “isolated nucleic acid” or “purified nucleic acid” alsorefer to a DNA molecules which comprises a coding region for a humanizedRAMP1 protein that has been purified away from other cellularcomponents. Thus, a humanized RAMP1 DNA preparation that issubstantially free from other nucleic acids will contain, as a percentof its total nucleic acid, no more than 10%, preferably no more than 5%,more preferably no more than 1%, and even more preferably no more than0.1%, of non-humanized RAMP1 nucleic acid molecules. Whether a givenhumanized RAMP1 preparation is substantially free from other nucleicacids can be determined by such conventional techniques of assessingnucleic acid purity as, e.g., agarose gel electrophoresis combined withappropriate staining methods, e.g., ethidium bromide staining, or bysequencing.

As used herein, “substantially free from other proteins” or“substantially purified” means at least 90%, preferably 95%, morepreferably 99%, and even more preferably 99.9%, free of other proteins.Thus, a humanized RAMP1 protein preparation that is substantially freefrom other proteins will contain, as a percent of its total protein, nomore than 10%, preferably no more than 5%, more preferably no more than1%, and even more preferably no more than 0.1%, of humanized RAMP1proteins. Whether a given humanized RAMP1 protein preparation issubstantially free from other proteins can be determined by suchconventional techniques of assessing protein purity as, e.g., sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combinedwith appropriate detection methods, e.g., silver staining orimmunoblotting. As used interchangeably with the terms “substantiallyfree from other proteins” or “substantially purified”, the terms“isolated humanized RAMP1 protein” or “purified humanized RAMP1 protein”also refer to humanized RAMP1 protein that has been isolated from anatural source. Use of the term “isolated” or “purified” indicates thathumanized RAMP1 protein has been removed from its normal cellularenvironment. Thus, an isolated humanized RAMP1 protein may be in acell-free solution or placed in a different cellular environment fromthat in which it occurs naturally. The term isolated does not imply thatan isolated humanized RAMP1 protein is the only protein present, butinstead means that an isolated humanized RAMP1 protein is substantiallyfree of other proteins and non-amino acid material (e.g., nucleic acids,lipids, carbohydrates) naturally associated with the humanized RAMP1protein in vivo. Thus, a humanized RAMP1 protein that is recombinantlyexpressed in a prokaryotic or eukaryotic cell and substantially purifiedfrom this host cell which does not naturally (i.e., withoutintervention) express this RAMP1 protein is of course “isolatedhumanized RAMP1 protein” under any circumstances referred to herein. Asnoted above, a humanized RAMP1 protein preparation that is an isolatedor purified humanized RAMP1 protein will be substantially free fromother proteins will contain, as a percent of its total protein, no morethan 10%, preferably no more than 5%, more preferably no more than 1%,and even more preferably no more than 0.1%, of non-humanized RAMP1proteins.

As used interchangeably herein, “functional equivalent” or “biologicallyactive equivalent” means a protein which does not have exactly the sameamino acid sequence as naturally occurring or humanized RAMP1, due toalternative splicing, deletions, mutations, substitutions, or additions,but retains substantially the same biological activity as the respectivenaturally occurring or humanized RAMP1. Such functional equivalents willhave significant amino acid sequence identity with naturally occurringor humanized RAMP1, especially with the presence of the “humanizing”tryptophan codon at amino acid residue 74.

As used herein, the term “functional” is used to describe a gene orprotein that, when present in a cell or in vitro system, performsnormally as if in a native or unaltered condition or environment.Therefore, a gene which is not functional (i.e., “non-functional”,“disrupted”, “altered”, or the like) will encode a protein which doesnot function as a wild type, native or non-altered protein, or encodesno protein at all. Such a non-functional gene may be the product of ahomologous recombination event as described herein, where anon-functional gene is targeted specifically to the region of the targetchromosome which contains a functional form of the gene, resulting in a“knock-out” of the wild type or native gene.

As used herein, a “modulator” is a compound that causes a change in theexpression or activity of a mammalian CGRP receptor, such as a human orhumanized CGRP receptor, or causes a change in the effect of theinteraction of the respective receptor with its ligand(s), or otherprotein(s), such as an antagonist or agonist.

As used herein, “rodent” relates to a species which is a member of theorder Rodentia, having a single pair of upper and lower incisors forgnawing, wherein the teeth grow continuously and a gap is evidentbetween the incisors and grinding molars. Preferred examples include forgeneration of transgenic animals include, but are not limited to, Rattusnorvegicus, Rattus rattus, and Mus musculus.

As used herein, “rat” relates to animals which from the point ofsystemic zoology belong to the genus Rattus. The transgenic animals ofthe present invention may be generated from any species of the genusRattus, including but not limited to Rattus norvegicus and Rattusrattus.

As used herein, “mouse” relates to animals which from the point ofsystemic zoology belong to the genus Mus. The transgenic animals of thepresent invention may be generated from any species of the genus Mus,such as the house mouse, Mus musculus.

As used herein, “cynomolgous” or “cyno” refers to a non-human primatealso referred to as a macaque, from the genus Macaca, such as but notlimited to Macaca cynomolgus.

As used herein, “marmoset” is known to include non-human primates whichpossess soft fur and claws (instead of nails) on all digits except thegreat toe, belonging to the family Callithricidae.

As used herein, “pig” is interchangeable with the term “porcine.”As usedherein, the term “mammalian” will refer to any mammal, including a humanbeing, except in the context of utilizing a—mammalian—RAMP1 sequence togenerate a—humanized—RAMP1 protein. In that context, of course, thehuman sequence is meant to be excluded.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the amino acid alignment of human, rat and mouse wild typeRAMP1 protein sequences. Amino acid residue 74, underlined and initalics, is the target amino acid for humanization of mammalian RAMP1protein sequences such as the mouse and rat sequence.

FIG. 2 shows the chemical structure of several CGRP antagonists,BIBN4096BS, Compound 1 and Compound 2.

FIG. 3 shows the constructed RAMP1 Chimeras and RAMP1 Mutagenesis.Chimera 1 was constructed by replacing the first 66 amino acids of ratRAMP1 with the human sequence. Chimera 2 was generated in a similarfashion by replacing the first 112 amino acids of rat RAMP1 with thosefrom human RAMP1. Hashed regions indicate human RAMP1 sequence; theremaining unfilled areas represent rat peptide sequence. Mutagenesis ofrat RAMP1 at position 74 produced a single RAMP1 point mutant.

FIG. 4 shows the alignment of amino acids 66–112 of RAMP1 from human,marmoset, rat, mouse and pig. A partial marmoset RAMP1 clone wasgenerated as described in Example 1.

FIG. 5 shows Western blotting analysis of rCRLR co-expressed withrRAMP1(rat RAMP1) and hRAMP1 (human RAMP1). The membranes from thecompetitive binding experiments, including rCRLR transfected with emptyvector (pcDNA3.1), were treated with Peptide-N-Glycosidase F (F),Endoglycosidase F1 (F1), or no enzyme. Samples were separated bySDS-PAGE, followed by western blot analysis with anti-rat CRLRantibodies.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an isolated or purified nucleic acidmolecule (polynucleotide) which encodes a humanized version of acalcitonin gene-related peptide (CGRP) receptor, which comprises theG-protein coupled receptor calcitonin-receptor-like receptor (CRLR) andthe receptor-activity-modifying protein-1 (RAMP1). More specifically,the present invention relates to isolated or purified vertebrate, andpreferably mammalian, nucleic acid molecules which encode derivative,humanized versions of the CGRP receptor, namely via DNA molecules whichencode chimeric, hybrid or mutant derivatives of a mammalian RAMP1sequence, which are shown herein to be responsible for the“humanization” of the CGRP receptor upon association with a vertebrate(and again, preferably a mammalian) CRLR receptor protein. The CRLR andRAMP1 DNA molecules disclosed herein may be co-transfected into a hostcell of choice wherein the recombinant host cell provides a source forsubstantial levels of an expressed functional, humanized version of aCGRP receptor. Therefore, these recombinantly expressed humanized CGRPreceptor proteins form a receptor complex in which small molecule CGRPreceptor antagonists display potency similar to that for a “wild type”human CGRP receptor. Such mutant receptors will be useful in cell basedassays, receptor binding assays and/or radioligand binding assays, and,as noted below, in the generation of transgenic animals which providefor this humanized CGRP receptor activity.

To this end, a particularly preferred aspect of the present inventionwhich is afforded only in view of this specification is the generationof non-human animal cells, non-human transgenic animals, such asfounders and littermates, especially transgenic “knock-in” animals,wherein the endogenous gene encoding RAMP1 has been engineered (i.e.,“humanized”) to provide for a CGRP receptor pharmacological profilesimilar to human CGRP receptor. Such non-human transgenic animals willpreferably provide for an altered genotype (endogenous CRLR and“humanized” RAMP1), which will provide for a phenotype whereby thepharmacological profile of the non-human transgenic animal in regard tomodulators of CGRP will mimic the human form of CGRP receptor. Variousnon-human transgenic animals may be contemplated in view of the findingdisclosed herein that alteration of a single amino acid residue in anon-human RAMP1 sequence (such as rat, mouse and pig, as shown herein,as well as additional species, such as cyno and canine) results in a“humanized” version of RAMP1 when complexed with a mammalian version ofCRLR. In other words, the species-specific pharmacology of knownantagonists is shown herein to be localized to the region at or aroundamino acid residue 74 of human RAMP1 (a tryptophan residue), such thatnon-human RAMP1 forms may be generated and used to generate transgenicanimals which express the humanized version along with or instead of theendogenous RAMP1 protein.

The present invention therefore relates to isolated or purified nucleicacid molecules which encode a chimeric, hybrid and/or mutant version ofa RAMP1 protein where such a protein is functional (i.e., whenco-expressed with CRLR will exhibit predicted pharmacologicalproperties), and furthermore wherein such a protein is humanized byvirtue of altering the amino acid that corresponds to human amino acidresidue 74 to a tryptophan residue. Such a nucleic acid molecule is partof the present invention whether it encodes a chimeric, hybrid orvarious mutant protein, so long as amino acid 74 has been altered fromits native residue to the human residue, namely tryptophan.

The present invention further relates to isolated or purified nucleicacid molecules which encode a chimeric, hybrid and/or mutant version ofa RAMP1 protein, wherein such a derivative RAMP1 protein comprises therespective amino acid sequence at least from about amino acid 1 to aminoacid 65 and from about amino acid 113 to about amino acid 148, whereinthe region corresponding from about amino acid 66 to amino acid 112 isat least partially derived from the human RAMP1 coding region. Such DNAmolecules will encode “humanized” RAMP1 proteins which, whenco-expressed with a CRLR gene, or functional derivative thereof, willresult in a CGRP receptor which mimics human CGRP receptorpharmacological properties.

The present invention further relates to isolated or purified nucleicacid molecules which encode a chimeric, hybrid and/or mutant version ofa RAMP1 protein, wherein such a derivative RAMP1 protein at leastcomprises a nucleotide change which results in an alteration of aminoacid residue 74 to a tryptophan residue, which results in a humanizedform of RAMP1. To this end, a specific embodiment of the presentinvention relates to an isolated or purified nucleic acid molecule fromrat wherein the codon for amino acid residue 74 is altered from a lysineresidue to a tryptophan residue. Another specific embodiment of thepresent invention relates to an isolated or purified nucleic acidmolecule from mouse wherein the codon for amino acid residue 74 isaltered from a lysine residue to a tryptophan residue. Yet anotherspecific embodiment of the present invention relates to an isolated orpurified nucleic acid molecule from cynomolgous wherein the codon forthe amino acid residue corresponding to human residue 74 is altered froma cysteine residue to a tryptophan residue (i.e., a “C74W” mutant).Still another specific embodiment of the present invention relates to anisolated or purified nucleic acid molecule from porcine (pig) whereinthe codon for the amino acid residue corresponding to human residue 74is altered from a arginine residue to a tryptophan residue (i.e., a“R74W” mutant). Therefore, the present invention further relates to anisolated nucleic acid molecule (polynucleotide) which encodes mRNA whichexpresses a humanized RAMP1 protein, this DNA molecule comprising thenucleotide sequence disclosed herein in Table 1 and listed as SEQ IDNO:1 (rat), SEQ ID NO:3 (mouse), SEQ ID NO:5 (a partial sequence fromcyno) and SEQ ID NO:7 (a partial sequence from porcine (pig)). Table 1discloses the nucleotide and predicted amino acid sequences of thesevarious mammalian RAMP1 sequences which, when expressed as a full lengthRAMP1 protein, correspond to a “humanized” form of RAMP1.

TABLE 1 Rat K74W RAMP1 Nucleotide Sequence ATGGCCCCCG GCCTGCGGGGCCTCCCGCGG CGCGGCCTCT GGCTGCTGCT GGCTCATCAT (SEQ ID NO:1) CTCTTCATGGTCACTGCCTG CCGGGACCCT GACTATGGTA CTCTCATCCA GGAGCTGTGT CTCAGCCGCTTCAAAGAGGA CATGGAGACC ATAGGGAAGA CTCTGTGGTG TGACTCGGGA AAGACCATAGGGAGCTATGG GGAGCTCACT CACTGCACCT GGCTCGTGGC AAACAAGATT GGCTGTTTCTGGCCCAATCC GGAAGTGGAC AAGTTCTTCA TTGCTGTCCA CCACCGCTAC TTCAGCAAGTGCCCAGTCTC GGGCAGGGCC CTGCGGGACC CTCCCAACAG CATCCTCTGC CCTTTCATTGTGCTCCCCAT TACGGTCACA CTGCTCATGA CTGCCCTGGT GGTCTGGAGG AGCAAGCGCACAGAGGGCAT CGTGTAG Rat K74W RAMP1 Amino Acid Sequence MAPGLRGLPRRGLWLLLAHH LFMVTACRDP DYGTLIQELC LSRFKEDMET IGKTLWCDWG (SEQ ID NO:2)KTIGSYGELT HCTWLVANKI GCFWPNPEVD KFFIAVHHRY FSKCPVSGRA LRDPPNSILCPFIVLPITVT LLMTALVVWR SKRTEGIV Mouse K74W RAMP1 Nucleotide SequenceATGGCCCCGG GCCTGCGGGG CCTCCCGCGG TGCGGCCTCT GGCTGCTGCT GGCTCACCAT (SEQID NO:3) CTCTTCATGG TCACTGCCTG CCGGGACCCT GACTATGGGA CTCTCATCCAGGAGCTGTGC CTCAGCCGCT TCAAGGAGAA CATGGAGACT ATTGGGAAGA CGCTATGGTGTGACTGGGGA AAGACCATAC AGAGCTATGG GGAGCTCACT TACTGCACCT GGCACGTGGCGCACACGATT GGCTGTTTCT GGCCCAATCC GGAAGTGGAC AGATTCTTCA TCGCTGTCCACCATCGATAC TTCAGCAAGT GCCCCATCTC GGGCAGGGCC CTGCGGGACC CTCCCAACAGCATCCTCTGC CCTTTCATTG CGCTCCCCAT TACGGTCACG CTGCTCATGA CTGCACTGGTGGTCTGGAGG AGCAAGCGCA CAGAGGGCAT CGTGTAG Mouse K74W RAMP1 Amino AcidSequence MAPGLRGLPR CGLWLLLAHH LFMVTACRDP DYGTLIQELC LSRFKENMETIGKTLWCDWG (SEQ ID NO:4) KTIQSYGELT YCTWHVAHTI GCFWPNPEVD RFFIAVHHRYFSKCPISGRA LRDPPNSILC PFIALPITVT LLMTALVVWR SKRTEGIV Cynomolgous RAMP1Nucleotide Sequence (C74W RAMP1; partial) GTGCCCTCCT CCAGGAGCTCTGCCTCACCC AGTTCCAGGT AGACATGGAG GCCGTCGGGG (SEQ ID NO:5) AGACGCTGTGGTGTGACTGG GGCAGGACCA TCGGGAGCTA CAGGGAGCTG GCCGACTGCA CCTGGCACATGGCGGAGAAG CTAGGCTGCT TCTGGCCCAA CGCAGAGGTG GACAGGTTCT TCCTGGCAGTGCACGGGCAC TACTTCAGGG CCTGCCCCAT CTCAGGCAGG GCCGTGCGGG ACCCGCCTGG CAGCGCynomolgous RAMP1 Amino Acid Sequence (C74W RAMP1; partial) ALLQELCLTQFQVDMEAVGE TLWCDWGRTI GSYRELADCT WHMAEKLGCF WPNAEVDRFF (SEQ ID NO:6)LAVHGHYFRA CPISGRAVRD PPGS Porcine (Pig) RAMP1 Nucleotide Sequence (R74WRAMP1; partial) AGGACCATCA GGAGCTATAA AGACCTCTCA GACTGCACCT GGCTCGTGGCGCAAAGGCTG (SEQ ID NO:7) GACTGCTTCT GGCCCAACGC GGCGGTGGAC AAGTTCTTCCTGGGAGTCCA CCAGCAGTAC TTCAGAAACT GCCCCGTCTC CGGCAGGGCC TTGCAGGACCCGCCCAGCAG CGTCCTCTGC CCCTTCATCG TCGTCCCCAT CCTGGCGACC CTGCTCATGACCGCACTGGT GGTCTGGCAG Porcine (Pig) RAMP1 Amino Acid Sequence (R74WRAMP1; partial) RTIRSYKDLS DCTWLVAQRL DCFWPNAAVD KFFLGVHQQY FRNCPVSGRALQDPPSSVLC (SEQ ID NO:8) PFIVVPILAT LLMTALVVWQ

The present invention also relates to biologically active fragments ormutants of SEQ ID NOs:1, 3, 5 and 7 which encode mRNA expressing ahumanized RAMP1 protein. Any such biologically active fragment and/ormutant will encode either a protein or protein fragment which at leastsubstantially mimics the pharmacological properties of human RAMP1,including but not limited to the humanized RAMP1 proteins as set forthin SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8, with SIDs 6and 8 representing partial sequences which span the region manipulatedfor humanization of the respective RAMP1 protein. Any suchpolynucleotide includes but is not necessarily limited to chimericconstructs (including but not limited to the exemplified chimericconstructs described herein), hybrid constructs, nucleotidesubstitutions, deletions, additions, amino-terminal truncations andcarboxy-terminal truncations such that these mutations encode mRNA whichmay co-express a functional humanized RAMP1 with a mammalian CRLRprotein in a eukaryotic cell so as to be useful for screening foragonists and/or antagonists of CGRP activity. To this end, preferredaspects of this portion of the present invention are disclosed in Table1 as SEQ ID NOs:1; 3 and 5, all of which encode a humanized version ofRAMP1.

The isolated nucleic acid molecules of the present invention may includea deoxyribonucleic acid molecule (DNA), such as genomic DNA andcomplementary DNA (cDNA), which may be single (coding or noncodingstrand) or double stranded, as well as synthetic DNA, such as asynthesized, single stranded polynucleotide. The isolated nucleic acidmolecule of the present invention may also include a ribonucleic acidmolecule (RNA).

The present invention also relates to recombinant vectors andrecombinant host cells, both prokaryotic and eukaryotic, which containthe nucleic acid molecules disclosed throughout this specification andwhich encode a humanized version of a CGRP receptor and associatedfragment thereof, substantially purified forms of associated humanizedversion of a CGRP receptor, recombinant membrane fractions comprisingthese proteins (e.g., active CGRP receptors comprising CRLR andhumanized RAMP1 proteins), associated mutant proteins, and methodsassociated with identifying compounds which specifically modulated humanCGRP receptor utilizing the humanized version of RAMP1 in variousassays.

The present invention also relates to a substantially purified form of ahumanized RAMP1 protein, which comprises the amino acid sequencedisclosed in Table 1 (e.g., SEQ ID NOs:2, 4, 6 and 8). The inventionfurther relates to a humanized RAMP1 protein which consists of the aminoacid sequence disclosed in Table 1 (e.g., SEQ ID NOs:2, 4, 6 and 8). Asnoted herein, while vertebrate sequences are within the scope of theinvention, mammalian sequences, including but not limited to thoseexemplified herein, are preferred.

Another preferred aspect of the present invention relates to asubstantially purified, fully processed (including proteolyticprocessing, glycosylation and/or phosphorylation), mature humanizedRAMP1 protein obtained from a recombinant host cell containing a DNAexpression vector comprising nucleotide sequence as set forth in SEQ IDNOs: 1, 3, 5 and 7 which express the respective humanized RAMP1 protein.It is especially preferred that the recombinant host cell be aeukaryotic host cell, such as a mammalian cell line.

Another aspect of the present invention relates to a substantiallypurified membrane preparation, partially purified membrane preparation,or cell lysate which has been obtained from a recombinant host celltransformed or transfected with a DNA expression vector which comprisesand appropriately expresses a complete open reading frame as set forth,for example, in SEQ ID NOs: 1, 3, 5 and 7, which results in a functionalform of the respective humanized RAMP1 protein. These recombinantmembranes will comprise humanized RAMP1 proteins such as those disclosedin Table 1 (i.e., SEQ ID NOs: 2, 4, 6 and 8), or additional equivalentswhich results in a humanized form of RAMP1, namely mammalian RAMP1proteins wherein the amino acid residue corresponding to human aminoacid residue 74 has been altered to code for a tryptophan residue.

A preferred aspect of this portion of the present invention relates to asubstantially purified membrane preparation, partially purified membranepreparation, or cell lysate which has been obtained from a recombinanthost cell transformed or transfected with a DNA expression vector whichcomprises and appropriately expresses a humanized RAMP1 protein asdescribed throughout this specification, in conjunction with a DNAexpression vector which comprises and appropriately expresses amammalian CRLR GPCR protein. Examples of mammalian nucleotide sequenceswhich may be utilized for such a purpose included but are not limited tothe human, rat and mouse nucleic acid molecules disclosed in Table 2 andset forth as SEQ ID NOs: 7, 9, and 11, which results in a functionalform of a mammalian CRLR GPCR which, when co-expressed with a humanizedRAMP1 protein, will be useful to screen for modulators which effect thehuman CGRP receptor. The subcellular membrane fractions and/ormembrane-containing cell lysates from the recombinant host cells (bothprokaryotic and eukaryotic as well as both stably and transientlytransformed cells) contain the functional and processed proteins encodedby the nucleic acid molecules disclosed herein. This recombinant-basedmembrane preparation will comprise a mammalian CRLR protein and ahumanized RAMP1 protein which is essentially free from contaminatingproteins. These subcellular membrane fractions will comprise “humanized”CGRP receptors which function efficiently for the screening ofmodulators (e.g., agonists and especially antagonists) of the human CGRPreceptor at levels which are at least similar to or possiblysubstantially above endogenous levels. Any such “humanized” CGRPreceptor-containing membrane preparation will be useful in variousassays to select for modulators of the respective CGRP receptor. Apreferred eukaryotic host cell of choice to express the CGRP receptor ofthe present invention is a mammalian cell line.

TABLE 2 Human CRLR Nucleotide Sequence ATGGAGAAAA AGTGTACCCT GTATTTTCTGGTTCTCTTGC CTTTTTTTAT GATTCTTGTT (SEQ ID NO:9) ACAGCAGAAT TAGAAGAGAGTCCTGAGGAC TCAATTCAGT TGGGAGTTAC TAGAAATAAA ATCATGACAG CTCAATATGAATGTTACCAA AAGATTATGC AAGACCCCAT TCAACAAGCA GAAGGCGTTT ACTGCAACAGAACCTGGGAT GGATGGCTCT GCTGGAACGA TGTTGCAGCA GGAACTGAAT CAATGCAGCTCTGCCCTGAT TACTTTCAGG ACTTTGATCC ATCAGAAAAA GTTACAAAGA TCTGTGACCAAGATGGAAAC TGGTTTAGAC ATCCAGCAAG CAACAGAACA TGGACAAATT ATACCCAGTGTAATGTTAAC ACCCACGAGA AAGTGAAGAC TGCACTAAAT TTGTTTTACC TGACCATAATTGGACACGGA TTGTCTATTG CATCACTGCT TATCTCGCTT CGCATATTCT TTTATTTCAAGAGCCTAAGT TGCCAAAGGA TTACCTTACA CAAAAATCTG TTCTTCTCAT TTGTTTGTAACTCTGTTGTA ACAATCATTC ACCTCACTGC AGTGGCCAAC AACCAGGCCT TAGTAGCCACAAATCCTGTT AGTTGCAAAG TGTCCCAGTT CATTCATCTT TACCTGATGG GCTGTAATTACTTTTGGATG CTCTGTGAAG CCATTTACCT ACACACACTC ATTGTGGTGG CCGTGTTTGCAGAGAAGCAA CATTTAATGT GGTATTATTT TCTTGGCTGG GGATTTCCAC TGATTCCTGCTTGTATACAT GCCATTGCTA GAAGCTTATA TTACAATGAC AATTGCTGGA TCAGTTCTGATACCCATCTC CTCTACATTA TCCATGGCCC AATTTGTGCT GCTTTACTGG TGAATCTTTTTTTCTTGTTA AATATTGTAC GCGTTCTCAT CACCAAGTTA AAAGTTACAC ACCAAGCGGAATCCAATCTG TACATGAAAG CTGTGAGAGC TACTCTTATC TTGGTGCCAT TGCTTGGCATTGAATTTGTG CTGATTCCAT GGCGACCTGA AGGAAAGATT GCAGAGGAGG TATATGACTACATCATGCAC ATCCTTATGC ACTTCCAGGG TCTTTTGGTC TCTACCATTT TCTGCTTCTTTAATGGAGAG GTTCAAGCAA TTCTGAGAAG AAACTGGAAT CAATACAAAA TCCAATTTGGAAACAGCTTT TCCAACTCAG AAGCTCTTCG TAGTGCGTCT TACACAGTGT CAACAATCAGTGATGGTCCA GGTTATAGTC ATGACTGTCC TAGTGAACAC TTAAATGGAA AAAGCATCCATGATATTGAA AATGTTCTCT TAAAACCAGA AAATTTATAT AATTGA Human CRLR Amino AcidSequence MEKKCTLYFL VLLPFFMILV TAELEESPED SIQLGVTRNK IMTAQYECYQ (SEQ IDNO:10) KIMQDPIQQA EGVYCNRTWD GWLCWNDVAA GTESMQLCPD YFQDFDPSEK VTKICDQDGNWFRHPASNRT WTNYTQCNVN THEKVKTALN LFYLTIIGHG LSIASLLISL GIFFYFKSLSCQRITLHKNL FFSFVCNSVV TIIHLTAVAN NQALVATNPV SCKVSQFIHL YLMGCNYFWMLCEGIYLHTL IVVAVFAEKQ HLMWYYFLGW GFPLIPACIH AIARSLYYND NCWISSDTHLLYIIHGPICA ALLVNLFFLL NIVRVLITKL KVTHQAESNL YMKAVRATLI LVPLLGIEFVLIPWRPEGKI AEEVYDYIMH ILMHFQGLLV STIFCFFNGE VQAILRRNWN QYKIQFGNSFSNSEALRSAS YTVSTISDGP GYSHDCPSEH LNGKSIHDIE NVLLKPENLY N Rat CRLRNucleotide Sequence ATGGATAAAA AGTGTACACT TTGTTTTCTG TTTCTCTTGCTTCTTAATAT GGCTCTCATC (SEQ ID NO:11) GCAGCAGAGT CGGAAGAAGG CGCGAACCAAACAGACTTGG GAGTCACTAG GAACAAGATC ATGACGGCTC AGTATGAATG TTACCAAAAGATCATGCAGG ATCCCATTCA ACAAGGAGAA GGCCTTTACT GCAACAGAAC CTGGGACGGATGGCTATGCT GGAATGACGT TGCAGCAGGA ACCGAGTCAA TGCAGTACTG CCCTGATTACTTTCAAGATT TTGATCCTTC AGAGAAGGTT ACAAAGATCT GTGACCAAGA TGGAAACTGGTTCAGACATC CAGATAGTAA CAGGACATGG ACAAACTACA CCTTGTGTAA CAACAGCACGCATGAGAAAG TGAAGACAGC ACTGAATTTG TTCTACCTAA CTATAATTGG ACATGGATTATCTATTGCCT CTCTGATCAT CTCACTCATC ATATTTTTTT ATTTCAAGAG CCTAAGTTGCCAACGGATTA CATTGCATAA AAACCTGTTC TTTTCATTTG TTTGTAATTC GATTGTGACAATCATTCACC TCACGGCAGT GGCCAATAAC CAGGCCTTAG TGGCCACAAA TCCTGTGAGCTGCAAGGTGT CCCAGTTCAT TCATCTTTAC CTGATGGGCT GTAACTACTT TTGGATGCTCTGTGAAGGCA TTTACCTGCA CACACTCATT GTGGTGGCTG TGTTTGCAGA GAAGCAGCACTTGATGTGGT ATTATTTTCT TGGCTGGGGG TTTCCTCTGC TTCCTGCCTG CATCCATGCCATCGCCAGAA GCTTGTATTA CAATGACAAC TGCTGGATCA GCTCAGACAC TCATCTCCTCTACATCATCC ATGGTCCCAT TTGTGCTGCT TTACTGGTAA ATCTCTTTTT CCTATTAAATATTGTACGTG TTCTCATCAC CAAGTTGAAA GTTACACACC AAGCAGAATC CAATCTCTACATGAAAGCTG TAAGAGCCAC TCTCATCTTG GTACCACTAC TTGGCATTGA ATTTGTGCTTTTTCCATGGC GGCCTGAAGG AAAGGTTGCT GAGGAGGTGT ATGACTATGT CATGCACATTCTCATGCACT ATCAGGGTCT TTTGGTGTCT ACAATTTTCT GCTTCTTTAA CGGAGAGGTTCAAGCAATTC TGAGAAGAAA TTGGAACCAG TATAAAATCC AATTTGGCAA TGGCTTTTCCCACTCTGATG CTCTCCGCAG CGCATCCTAT ACGGTGTCAA CAATCAGCGA TGTGCAGGGGTACAGCCACG ACTGCCCCAC TGAACACTTA AATGGAAAAA GCATCCAGGA TATTGAAAATGTTGCCTTAA AACCAGAAAA AATGTATGAT CTAGTGATGT GA Rat CRLR Amino AcidSequence MMDKKCTLCF LFLLLLNMAL IAAESEEGAN QTDLGVTRNK IMTAQYECYQ (SEQ IDNO:12) KIMQDPIQQG EGLYCNRTWD GWLCWNDVAA GTESMQYCPD YFQDFDPSEK VTKICDQDGNWFRHPDSNRT WTNYTLCNNS THEKVKTALN LFYLTIIGHG LSIASLIISL IIFFYFKSLSCQRITLHKNL FFSFVCNSIV TIIHLTAVAN NQALVATNPV SCKVSQFIHL YLMGCNYFWMLCEGIYLHTL IVVAVFAEKQ HLMWYYFLGW GFPLLPACIH AIARSLYYND NCWISSDTHLLYIIHGPICA ALLVNLFFLL NIVRVLITKL KVTHQAESNL YMKAVRATLI LVPLLGIEFVLFPWRPEGKV AEEVYDYVMH ILMHYQGLLV STIFCFFNGE VQAILRRNWN QYKIQFGNGFSHSDALRSAS YTVSTISDVQ GYSHDCPTEH LNGKSIQDIE NVALKPEKMY DLVM Mouse CRLRNucleotide Sequence ATGGATAAAA AGCATATACT ATGTTTTCTG GTTCTCTTGCCTCTTAATAT GGCTCTCATC (SEQ ID NO:13) TCAGCAGAGT CGGAAGAAGG CGTGAACCAAACAGACTTGG GAGTCACTAG AAACAAGATC ATGACGGCTC AATATGAATG TTACCAGAAGATCATGCAGG ACCCCATTCA ACAAGCAGAA GGCCTTTACT GCAATAGGAC CTGGGACGGATGGCTATGCT GGAATGACGT TGCAGCAGGG ACGGAATCAA TGCAGTACTG CCCTGACTATTTTCAGGATT TTGATCCTTC AGAGAAGGTT ACAAAGATCT GTGACCAAGA TGGACACTGGTTTCGGCATC CGGATAGTAA TAGAACATGG ACCAACTACA CCCTGTGTAA TAACAGCACGCATGAGAAAG TGAAGACAGC CCTGAATCTG TTCTACCTAA CTATAATTGG ACATGGATTATCTATTGCAT CTCTGATCAT CTCTCTCATC ATATTTTTTT ACTTCAAGAG CCTAAGTTGCCAACGGATCA CATTGCATAA AAACCTGTTC TTTTCATTTA TTTGTAATTC AATTGTAACAATCATCCACC TCACGGCAGT GGCCAATAAC CAGGCCTTAG TGGCCACAAA TCCTGTGAGCTGCAAAGTGT CTCAGTTTAT CCATCTCTAC CTGATGGGCT GTAACTACTT CTGGATGCTCTGTGAAGGCG TTTACCTGCA CACACTCATC GTGGTGGCTG TGTTTGCGGA GAAGCAGCACTTGATGTGGT ATTATTTTCT CGGCTGGGGG TTTCCTCTGC TTCCTGCCTG CATCCACGCCATTGCCAGAA GCTTGTATTA CAACGACAAT TGCTCGATCA GCTCAGACAC TCATCTCCTCTACATTATCC ATGGTCCGAT TTGTGCTGCT TTGTTGGTAA ATCTCTTTTT CCTATTAAATATTGTACGTG TTCTCATCAC CAAGTTGAAA GTTACACACC AAGTGGAATC CAATCTCTACATGAAAGCCG TAAGAGCTAC TCTCATCTTG GTACCACTAC TTGGCATTGA ATTTGTGCTTTTTCCGTGGC GGCCTGAAGG AAAGGTTGCA GAGGAGGTGT ATGACTATGT CATGCACATTTTGATGCACT TTCAGGGTCT TTTGGTGGCT ACTATTTTCT GCTTCTTTAA TGGAGAGGTTCAAGCAATTC TGAGAAGAAA TTGGAACCAG TATAAAATCC AATTTGGAAA TGGCTTTTCCCACTCTGATG CTCTCCGCAG TGCATCCTAC ACAGTGTCAA CAATCAGTGA CATGCAAGGGTACAGCCATG ACTGCCCCAC TGAACACTTA AATGGAAAAA GCATCCAGGA TATTGAAAATGTTGCCTTAA AATCAGAAAA TATGTATGAT CTAGTGATGT GA Mouse CRLR Amino AcidSequence MDKKHILCFL VLLPLNMALI SAESEEGVNQ TDLGVTRNKI MTAQYECYQK (SEQ IDNO:14) IMQDPIQQAE GLYCNRTWDG WLCWNDVAAG TESMQYCPDY FQDFDPSEKV TKICDQDGHWFRHPDSNRTW TNYTLCNNST HEKVKTALNL FYLTIIGHGL SIASLIISLI IFFYFKSLSCQRITLHKNLF FSFICNSIVT IIHLTAVANN QALVATNPVS CKVSQFIHLY LMGCNYFWMLCEGVYLHTLI VVAVFAEKQH LMWYYFLGWG FPLLPACIHA IARSLYYNDN CWISSDTHLLYIIHGPICAA LLVNLFFLLN IVRVLITKLK VTHQVESNLY MKAVRATLIL VPLLGIEFVLFPWRPEGKVA EEVYDYVMHI LMHFQGLLVA TIFCFFNGEV QAILRRNWNQ YKIQFGNGFSHSDALRSASY TVSTISDMQG YSHDCPTEHL NGKSIQDIEN VALKSENMYD LVM

The present invention also relates to biologically active fragmentsand/or mutants of a humanized RAMP1 protein, comprising the amino acidsequence as set forth in SEQ ID NOs: 2, 4, 6 or 8, including but notnecessarily limited to amino acid substitutions, deletions, additions,amino terminal truncations and carboxy-terminal truncations such thatthese mutations provide for proteins or protein fragments of diagnostic,therapeutic or prophylactic use and would be useful for screening forselective modulators, including but not limited to agonists and/orantagonists for human CGRP receptor pharmacology.

A preferred aspect of the present invention is disclosed in Table 1 asSEQ ID NOs:2, 4, 6 and 8, respective amino acid sequences which aremammalian RAMP1 proteins, or portions thereof, which have been“humanized” solely by altering amino acid residue 74 to a tryptophan(“Trp” or “W”) residue. As noted above, co-expression of a humanizedRAMP1 protein of the present invention along with a mammalian CRLRprotein will be useful in screening for antagonists of the CGRPreceptor.

The present invention also relates to polyclonal and monoclonalantibodies raised against forms of humanized RAMP1, a biologicallyactive fragment of humanized RAMP1, or a CGRP receptor complex whichcomprises a humanized RAMP1.

The present invention also relates to isolated nucleic acid moleculeswhich encode humanized RAMP1 fusion constructs (as well as thesubstantially purified protein expressed within and recovered from therespective host cell which houses the fusion construct, most likely inthe form of a DNA expression vector), including but not limited tofusion constructs which express a portion of humanized RAMP1 to variousmarkers, including but in no way limited to GFP (Green fluorescentprotein), the MYC epitope, GST, Fc, Flag, HA, and His-tag. Any suchfusion construct will comprise at least a portion of the RAMP1 openreading frame which encodes for the alteration at amino acid 74 to atryptophan residue, such that the respective fusion protein will exhibithuman-like pharmacological properties when complexed with a mammalianCRLR protein.

As noted above, the heterodimeric CGRP receptor requires co-expressionof calcitonin receptor-like receptor (CRLR) and an accessory proteincalled receptor activity modifying protein 1, or RAMP1. Several smallmolecule CGRP receptor antagonists have been shown to exhibit markedspecies selectivity, with >100-fold higher affinities for the human CGRPreceptor than for receptors from other species. It is shown herein thatspecies selectivity of CGRP modulators is determined exclusively byRAMP1. By constructing hybrid human/rat CRLR/RAMP1 receptors, it isdisclosed herein that co-expression of hCRLR with rRAMP1 produced ratreceptor pharmacology, and vice versa (h=human, r=rat, m=mouse).Moreover, with rat/human RAMP1 chimeras and site-directed mutants, it isfurther disclosed herein that a single amino acid at position 74 of theRAMP1 protein modulates the affinity of small molecule antagonists forCRLR/RAMP1. Co-expression of rCRLR with rK74W RAMP1 and mCRLR with mK74WRAMP1 increased the affinities of these antagonists by >100-fold,resulting in IC₅₀ values similar to those observed for the humanreceptor. Therefore, it is disclosed herein that the affinities of smallmolecule antagonists for the CGRP receptor are heavily influenced by thenature of amino acid 74 of RAMP1 and provide evidence that RAMP1participates in the antagonist binding sites.

It is shown herein that amino acid position 74 of RAMP1 is responsiblefor the observed species selectivity of several known antagonists of theCGRP receptor, suggesting that that the affinity of small moleculeantagonists can be affected by a single amino acid change and that theseantagonists may interact directly with RAMP1. The identification of asingle amino acid mutation that can convert the mouse CGRP receptor intoone that displays human-like pharmacology shows that a humanized CGRPreceptor mouse may be created by a “knock-in” strategy, whereinlysine-74 is replaced with tryptophan by various techniques well knownin the art. Such a humanized non-human transgenic animal (e.g., atransgenic mouse), will have utility in drug discovery and developmentprograms for in vivo pharmacological studies of CGRP receptorantagonists, as well as complementing marmoset as a suitable animalmodel for such studies.

To this end, the present invention relates to a transgenic non-humananimal, such as a founder animal or subsequent littermate, wherein bothalleles of the endogenous RAMP1 gene have been humanized, as well asheterozygous transgenic non-human animals wherein a single endogenousRAMP1 allele has been humanized and to non-human transgenic animalcomprising wild type endogenous RAMP1 alleles in addition to at leastone humanized RAMP1 allele stably integrated within the respectivetarget genome. To this end, the present invention relates to animalcells, non-human transgenic embryos, non-human transgenic animals andnon-human transgenic littermates which are homozygous for humanizedRAMP1 and whereby endogenous RAMP1 has been disrupted, namely byreplacement of the endogenous RAMP1 coding region, or portion thereof,by direct gene targeting within the respective target genome. Thepresent invention also extends to animal cells, non-human transgenicembryos, non-human transgenic animals (such as founder animals andtransgenic littermates) which are heterozygous for a functional RAMP1gene native to that animal. Namely, the heterozygosity referring the onefunctional, endogenous RAMP1 gene and one functional, humanized RAMP1gene. Also, the present invention relates to animal cells, non-humantransgenic embryos and non-human transgenic littermates having at leastone and possibly multiple humanized RAMP1 genes being randomly insertedwithin the target genome, such that both functional endogenous andhumanized RAMP1 proteins may be expressed. The transgenic animals of thepresent invention can be used in the study of the effect of modulators,especially antagonists, of the CGRP receptor. Such a transgenicnon-human animal will be especially useful for in vivo efficacy andreceptor occupancy studies for testing of CGRP receptor modulators,especially antagonists, for treatment of various disorders, includingbut not limited to migraine headaches, pain, menopausal hot flash,migraine prophylaxis, chronic tension type headache, cluster headache,neurogenic or chronic inflammation, gastrointestinal disorders, type 2diabetes and cardiovascular disorders (via agonizing the CGRP receptor).Generation of a genetically engineered mouse expressing a human-likemutant RAMP1 will result in a species in which small molecule CGRPreceptor antagonists display potency similar to that for the human CGRPreceptor. The non-human transgenic animal of the present invention mayalso provide cells for culture, for in vitro studies. Therefore, inparticular embodiments of the present invention, cell lines are producedand cells isolated from any of the animals produced in the stepsdescribed herein.

An aspect of this portion of the invention is a method to obtain ananimal wherein the endogenous RAMP1 gene native to the animal has beenreplaced by “knock-in” technology such that a humanized form of RAMP1has replaced the endogenous RAMP1 allele(s). A RAMP1 gene that naturallyoccurs in the animal is referred to as the native gene, endogenous geneand/or “wild-type” gene. It is preferred that expression of a non-nativeRAMP1 gene (e.g., a “humanized” RAMP1 gene) take place in a transgenicanimal in the absence of a native RAMP1 gene. Such a transgenic“knock-in” non-human animal (such as a transgenic mouse) will beespecially useful in animal studies to mimic pharmacology of the humanCGRP receptor while utilizing an endogenous CRLR gene and a “humanized”RAMP1 gene. The method includes providing a gene for a humanized form ofRAMP1 in the form of a transgene and targeting the transgene into achromosome of the animal at the place of the native RAMP1 gene or atanother chromosomal location. The transgene can be introduced into theembryonic stem cells by a variety of methods known in the art, includingelectroporation, microinjection, and lipofection. Cells carrying thetransgene can then be injected into blastocysts which are then implantedinto pseudopregnant animals. In alternate embodiments, thetransgene-targeted embryonic stem cells can be co-incubated withfertilized eggs or morulae followed by implantation into females. Aftergestation, the animals obtained are chimeric founder transgenic animals.The founder animals can be used in further embodiments to cross withwild-type animals to produce F1 animals heterozygous for the alteredRAMP1 gene. In further embodiments, these heterozygous animals can beinterbred to obtain the viable transgenic embryos whose somatic and germcells are homozygous for the altered, humanized RAMP1. In otherembodiments, the heterozygous animals can be used to produce cellslines. In preferred embodiments, the animals are mice or rat. Therefore,a preferred aspect of this portion of the present invention is atransgenic non-human animal which expresses a non-native, humanizedRAMP1 protein on a native RAMP1 null background. As noted above, theanimal can be heterozygous (i.e., having a different allelicrepresentation of a gene on each of a pair of chromosomes of a diploidgenome, such as native RAMP1/humanized RAMP1), homozygous (i.e., havingthe same representation of a gene on each of a pair of chromosomes of adiploid genome, such as humanized RAMP1/humanized RAMP1) for the alteredRAMP1 gene, hemizygous (i.e., having a gene represented on only one of apair of chromosomes of a diploid genome, preferably a humanized versionof RAMP1), or homozygous for the humanized RAMP1 gene. In preferredembodiments, the animal is a mouse or a rat, with mouse being especiallypreferred. In a further embodiment, the targeted or randomly insertedhumanized RAMP1 gene may be operably linked to a promoter. As usedherein, operably linked is used to denote a functional connectionbetween two elements whose orientation relevant to one another can vary.In this particular case, it is understood in the art that a promoter canbe operably linked to the coding sequence of a gene to direct theexpression of the coding sequence while placed at various distances fromthe coding sequence in a genetic construct. Further embodiments are celllines and cells derived from animals of this aspect of the invention.

The non-human transgenic animals of the present invention includenon-human mammalian species which are candidates for humanization,including but not limited to transgenic mice, transgenic rats, as wellas non-human primates which are candidates for RAMP1 humanization.Transgenic mice are preferred.

The present invention especially relates to analysis of the complexfunction(s) of the CGRP receptor. The native wild type gene isselectively replaced via targeted gene delivery or it resides within thesame genome by random integration of a humanized RAMP1 gene intotipotent ES cells and used to generate the transgenic mice of thepresent invention. Techniques are available to replace an endogenoushomologue or to randomly insert such a homologue into the endogenousgenomic background by using known targeted homologous recombination orrandom integration, respectively, to generated genotypic changes intochromosomal alleles. Therefore, as noted above, the present inventionrelates to diploid animal cells, non-human transgenic embryos, non-humantransgenic animals and non-human transgenic founders and/or transgeniclittermates which are heterozygous or homozygous for a disrupted RAMP1gene and/or insertion of a humanized RAMP1 gene. The cells, embryos andnon-human transgenic animals contain two chromosome alleles forhumanized RAMP1 wherein at least one of the wild type RAMP1 alleles ismutated such that less than wild-type levels of RAMP1 activity isproduced. The diploid cell, embryo or non-human transgenic animalhomozygous for a humanized RAMP1 gene, wherein a humanized RAMP1 genehas been targeted to replace the wild type allele, may show at leastfrom about 50%, and preferably about 100% reduction in wild type RAMP1activity (measured by the loss of “wild type” pharmacologicalcharacteristics of the endogenous CGRP receptor) and a concomitant CGRPreceptor activity which mimics human CGRP receptor pharmacology, ascompared to a wild type diploid cell. A diploid mouse cell, embryo ornon-human transgenic mouse generated herein which is heterozygous for adisrupted RAMP1 gene (i.e., wtRAMP1/humanized RAMP1) gene may show atleast from about 10% to about 100% reduction in endogenous RAMP1activity compared to a wild type diploid cell. It is within the purviewof the artisan of ordinary skill to use known molecular biologytechniques to measure the level of transcription, expression and/orfunctional CRLR/RAMP1 activity in mouse cell homozygous, heterozygous orhemizygous for a humanized RAMP1 gene. Therefore, the present inventionespecially relates to analysis of the complex function(s) of the CGRPreceptor by generating homozygous, heterozygous or hemizygous transgenicmice and studying how various potential modulators interact within thesemanipulated animals. In a preferred embodiment, the assay is performedby providing an animal of the present invention (especially a transgenicanimal wherein a humanized RAMP1 gene has replaced the endogenous RAMP1gene at both alleles), exposing the animal to a compound (preferably apotential antagonist of CGRP receptor activity), and measuring theeffect of said compound on biochemical and physiological responsesrelated to CGRP activity, or lack thereof. The measurement can becompared to these measurements in a genetically similar or identicalanimal that is not exposed to the compound.

As introduced above, a type of target cell for transgene introduction ispreferably the embryonic stem cell (ES), especially when generating atransgenic mouse, where culturing of ES cells has been particularlysuccessful. ES cells can be obtained from pre-implantation embryoscultured in vitro and fused with embryos (Evans et al., 1981, Nature292: 154–156; Bradley et al., 1984, Nature 309: 255–258; Gossler et al.,1986, Proc. Natl. Acad. Sci. USA 83: 9065–9069; and Robertson et al.,1986, Nature 322: 445–448). Transgenes can be efficiently introducedinto the ES cells by a variety of standard techniques such as DNAtransfection, microinjection, or by retrovirus-mediated transduction.The resultant transformed ES cells can thereafter be combined withblastocysts from a non-human animal. The introduced ES cells thereaftercolonize the embryo and contribute to the germ line of the resultingchimeric animal (Jaenisch, 1988, Science 240: 1468–1474). The use ofgene-targeted ES cells in the generation of gene-targeted transgenicmice was described in 1987 (Thomas et al., Cell 51:503–512, (1987)) andis reviewed elsewhere (Frohman et al., Cell 56:145–147 (1989); Capecchi,Trends in Genet. 5:70–76 (1989); Baribault et al., Mol. Biol. Med.6:481–492, (1989); Wagner, EMBO J. 9:3025–3032 (1990); Bradley et al.,Bio/Technology 10:534–539 (1992)). See also, U.S. Pat. No. 5,464,764,issued to Cappecchi and Thomas on Nov. 7, 1995; U.S. Pat. No. 5,789,215,issued to Berns et al on Aug. 4, 1998, both of which are herebyincorporated by reference). Therefore, techniques are available in theart to generate the transgenic animal cells, non-human transgenicembryos, non-human transgenic animals and non-human transgeniclittermates of the present invention. The methods for evaluating thetargeted recombination events as well as the resulting knockout mice arealso readily available and known in the art. Such methods include, butare not limited to DNA (Southern) hybridization to detect the targetedallele, polymerase chain reaction (PCR), polyacrylamide gelelectrophoresis (PAGE), in situ hybridization, RNA/Northernhybridization and Western blots to detect DNA, RNA and protein.

It is now well known in the art that various strategies are readilyavailable to the artisan to generated transgenic animals, such astransgenic “knock-in” animals. For example, BAC recombinationtechnologies, the following of which are expressly incorporated byreference in their entirety, include but are not limited to theteachings of Shizuya, et al., 1992, Proc. Natl. Acad. Sci. USA 89:8794–8797 (introduction of BAC vectors); Zhang et al., 1998, NatureGenetics 20: 123–128 and Muyrers, et al., 2001, Nucleic Acids Research27(6): 1555–1557 (modification of BAC clones via plasmid basedexpression of recA/recT proteins from the Rac phage or radα or radβ fromλ phage, respectively, for a review see also Muyrers et al. 2001, Trendsin Biochemical Sciences 26(5): 325–331); Yu et al. 2000, Proc. Natl.Acad. Sci. USA 97(11): 5978–5983 and Lee et al., 2001, Genomics 73:56–65 (use of a defective λ prophage to provide for radα or radβproteins to promote BAC-based recombination). These technologies allowfor the efficient engineering and manipulation of BAC clones to generatean appropriate targeting vector delivery to and recombination within EScells or harvested pronuclie. It is also known that techniques arereadily available that promote site specific recombination, allowing forprecise chromosome and transgene engineering. For a review of two wellknown systems, the FLP recombinase from yeast and Cre recombinase systemfrom bacteriophage P1, see Kilby et al., 1993, Trends Genetics 9:413–421, as well as U.S. Pat. Nos. 5,654,182; 5,677,177; and 5,885,836(FLP/frt) and U.S. Pat. No. 4,959317 (Cre/loxP), each U.S. patent whichis hereby incorporated by reference in their entirety. Therefore, thistechnology may be utilized to identify a RAMP1 genomic clone (such as amouse genomic clone), modifying such a genomic clone so as to humanizethe coding sequence (i.e., Lys to Trp at amino acid residue 74; where,for example, in generating a transgenic mouse, the only modificationrequired will be the mutagenesis of 2 nucleotides to change the Lysine(AAG) to a Tryptophan (TGG), which results in introduction of a BstNIrestriction site [from CCAAG to CCTGG], which is helpful for screeningpurposes) and to then deliver and stably incorporate, either byhomologous or non-homologous recombination, to an ES cell or pronucleus.To provide guidance in developing a humanized mouse “knock in” strategy,the mouse sequence consortium (MSC) database is queried with RAMP1nucleotide sequence. An initial search resulted in 2 mouse genomicsequence “hits” which were identified as mouse RAMP1. These 2 hitsencoded putative exons 2 and 3 of mouse RAMP1. Putative exons 2 and 3were found on a 712 bp fragment and a 1339 bp contig of 2 fragments,respectively. Putative exon 3 contains amino acid residue 74. Thisinformation can be utilized to design a probe for mouse BAC libraryscreening to obtain putative exon 3 and the surrounding intronicsequence for targeting vector construction. The genomic organizationappears to be conserved between human and mouse with intron/exon borderslocated at similar residues (Derst et al., 2000, Cytogenet Cell Genet90: 115–118).

It will be within the scope of the invention to submit screenedcompounds which show an in vitro modulation effect on humanized CGRPreceptor to in vivo analysis, preferably by administering the compoundof interest to either a transgenic or wild-type animal as describedherein to measure in vivo effects of the compound on this humanized CGRPreceptor and to further measure biological and physiological effects ofcompound administration on the non-human animal. These in vivo studiesmay be done either alone or in combination with a known RAMP1.

The transgenic non-human animal models as described herein will beuseful to screen any potential modulator of CGRP receptor activity(e.g., antagonists or agonists), including but not necessarily limitedto peptides, proteins, or non-proteinaceous organic or inorganicmolecules. To this end, the present invention relates to processes forthe production of the transgenic animals of the present invention andtheir offspring and their use for pharmacological testing. The inventionfurther relates to methods of determining the selectivity and activityof potential modulators (especially antagonists) of humanized CGRPreceptors expressed within transgenic animals of the present inventionby administering a test compound or compounds to the transgenic animaland measuring the effect of the compound on the activity of thehumanized CGRP receptor. To this end, the present invention relates tovarious occupancy assays which may be run in conjunction with thetransgenic non-human animals of the present invention.

As used and exemplified herein, a transgene is a genetic constructincluding a gene. The transgene of interest is incorporated into thetarget genome of the target cell, thus being introduced into their germcells and/or somatic cells such that it is stably incorporated and iscapable of carrying out a desired function. While a chromosome is thepreferred target for stable incorporation of a transgene into the targetanimal, the term “genome” refers to the entire DNA complement of anorganism, including nuclear DNA (chromosomal or extrachromosomal DNA) aswell as mitochondrial DNA, which is localized within the cytoplasm ofthe cell. Thus, the transgenic non-human animal of the present inventionwill stably incorporate one or more transgenes in either/or of the mousegerm cells or somatic cells (preferably both), such that the expressionof the transgene (e.g., a humanized form of mammalian RAMP1) achievesthe desired effect of presenting a specific receptor occupancy model formodulators of human RAMP1 as well as providing for an pharmacodynamicanimal model system to study the selectivity of test compounds tomodulate the human RAMP1 receptor. It is preferable to introduce thetransgene into a germ line cell, thereby conferring the ability totransfer the information to offspring. If offspring in fact possess someor all of the genetic information, then they, too, are transgenicanimals.

As used herein, the term “animal” may include all mammals, except thatwhen referring to transgenic animals, the use of this term excludeshumans. It also includes an individual animal in all stages ofdevelopment, including embryonic and fetal stages. A “transgenic animal”is an animal containing one or more cells bearing genetic informationreceived, directly or indirectly, by deliberate genetic manipulation ata subcellular level, such as by microinjection, targeted gene deliverysuch as by homologous recombination, or infection with recombinantvirus. As noted above, this introduced DNA molecule (i.e., transgene)can be integrated within a chromosome, or it can be extra-chromosomallyreplicating DNA.

As used herein in reference to transgenic animals of this invention, werefer to “transgenes” and “genes”. A gene is a nucleotide sequence thatencodes a protein, or structural RNA. The gene and/or transgene may alsoinclude genetic regulatory elements and/or structural elements known inthe art. As used and exemplified herein, a transgene is a geneticconstruct including a gene. The transgene is integrated into one or morechromosomes in the cells in an animal by methods known in the art. Onceintegrated, the transgene is carried in at least one place in thegenome, preferably a chromosome, of a transgenic animal.

As used herein, “founder” refers to a transgenic animal which developsfrom the microinjected egg. The founders are tested for expression of afunctional gene by any suitable assay of the gene product.

As used herein, the term “line” refers to animals that are directdescendants of one founder and bearing one transgene locus stablyintegrated into their germline.

As used herein, the term “inbred line” refers to animals which aregenetically identical at all endogenous loci. As used in the art, inbredlines may be used for including reproducibility from one animal to thenext, ability to transfer cells or tissue among animals, and the abilityto carry out defined genetic studies to identify the role of endogenousgenes. Such inbred lines may be developed from such lines wherein themice that are used for microinjection are members of established inbredstrains.

As used herein, the term “genotype” is the genetic constitution of anorganism.

As used herein, the term “phenotype” is a collection of morphological,physiological and/or biochemical traits possessed by a cell or organismthat results from the interaction of the genotype and the environment.Included in this definition of phenotype is a biochemical trait whereina non-native transgene has been introduced into the animal, thusaltering its the genotypic profile, and whereby expression of thistransgene(s) within the animal results in a new pharmacologicalselectivity to one or more chemical compounds, such a selectivity basedon functional expression of the transgene(s) of interest. To this end,the term “phenotypic expression” relates to the expression of atransgene or transgenes which results in the production of a product,e.g., a polypeptide or protein, or alters the expression of the zygote'sor the organism's natural phenotype.

The transgene of interest is incorporated into the target genome of thetarget cell, thus being introduced into their germ cells and/or somaticcells such that it is stably incorporated and is capable of carrying outa desired function. While a chromosome is the preferred target forstable incorporation of a transgene into the target animal, the term“genome” refers to the entire DNA complement of an organism, includingnuclear DNA (chromosomal or extrachromosomal DNA) as well asmitochondrial DNA, which is localized within the cytoplasm of the cell.Thus, as noted previously, the transgenic non-human animals of thepresent invention will stably incorporate one or more transgenes ineither/or of the animal's germ cells or somatic cells (preferably both),such that the expression of the transgene (e.g., a functional, humanizedversion of RAMP1) achieves the desired effect of presenting a specificreceptor occupancy model for modulators of a humanized CGRP receptor aswell as providing for an pharmacodynamic animal model system to studythe selectivity of test compounds to modulate a humanized CGRP receptorwhich comprises an endogenous CRLR protein and a humanized RAMP1protein. It is preferable to introduce the transgene into a germ linecell, thereby conferring the ability to transfer the information tooffspring. If offspring in fact possess some or all of the geneticinformation, then they, too, are transgenic animals.

As used herein, the term “animal” may include all mammals, except thatwhen referring to transgenic animals, the use of this term excludeshumans. It also includes an individual animal in all stages ofdevelopment, including embryonic and fetal stages. A “transgenic animal”is an animal containing one or more cells bearing genetic informationreceived, directly or indirectly, by deliberate genetic manipulation ata subcellular level, such as by microinjection, targeted gene deliverysuch as by homologous recombination, or infection with recombinantvirus. As noted above, this introduced DNA molecule (i.e., transgene)can be integrated within a chromosome, or it can be extra-chromosomallyreplicating DNA. In a preferred aspect of the present invention atargeted “knock-in” is performed whereby a humanized version of RAMP1 isinserted and replaces the endogenous RAMP1 coding region.

The degeneracy of the genetic code is such that, for all but two aminoacids, more than a single codon encodes a particular amino acid. Thisallows for the construction of synthetic DNA that encodes a humanizedRAMP1 protein where the nucleotide sequence of the synthetic DNA differssignificantly from the nucleotide sequences disclosed herein, asexemplification but not limitations, but still encodes a humanized RAMP1protein. Such synthetic DNAs are intended to be within the scope of thepresent invention. If it is desired to express such synthetic DNAs in aparticular host cell or organism, the codon usage of such synthetic DNAscan be adjusted to reflect the codon usage of that particular host, thusleading to higher levels of expression of the humanized RAMP1 protein inthe host. In other words, this redundancy in the various codons whichcode for specific amino acids is within the scope of the presentinvention. Therefore, this invention is also directed to those DNAsequences which encode RNA comprising alternative codons which code forthe eventual translation of the identical amino acid, as shown below:

-   A=Ala=Alanine: codons GCA, GCC, GCG, GCU-   C=Cys=Cysteine: codons UGC, UGU-   D=Asp=Aspartic acid: codons GAC, GAU-   E=Glu=Glutamic acid: codons GAA, GAG-   F=Phe=Phenylalanine: codons UUC, UUU-   G=Gly=Glycine: codons GGA, GGC, GGG, GGU-   H=His=Histidine: codons CAC, CAU-   I=Ile=Isoleucine: codons AUA, AUC, AUU-   K=Lys=Lysine: codons AAA, AAG-   L=Leu=Leucine: codons UUA, UUG, CUA, CUC, CUG, CUU-   M=Met=Methionine: codon AUG-   N=Asp=Asparagine: codons AAC, AAU-   P=Pro=Proline: codons CCA, CCC, CCG, CCU-   Q=Gln=Glutamine: codons CAA, CAG-   R=Arg=Arginine: codons AGA, AGG, CGA, CGC, CGG, CGU-   S=Ser=Serine: codons AGC, AGU, UCA, UCC, UCG, UCU-   T=Thr=Threonine: codons ACA, ACC, ACG, ACU-   V=Val=Valine: codons GUA, GUC, GUG, GUU-   W=Trp=Tryptophan: codon UGG-   Y=Tyr=Tyrosine: codons UAC, UAU    Therefore, the present invention discloses codon redundancy which    may result in differing DNA molecules expressing an identical    protein. For purposes of this specification, a sequence bearing one    or more replaced codons will be defined as a degenerate variation.    Another source of sequence variation may occur through RNA editing,    as discussed infra. Such RNA editing may result in another form of    codon redundancy, wherein a change in the open reading frame does    not result in an altered amino acid residue in the expressed    protein. Also included within the scope of this invention are    mutations either in the DNA sequence or the translated protein which    do not substantially alter the ultimate physical properties of the    expressed protein. For example, substitution of valine for leucine,    arginine for lysine, or asparagine for glutamine may not cause a    change in functionality of the polypeptide.

It is known that DNA sequences coding for a peptide may be altered so asto code for a peptide having properties that are different than those ofthe naturally occurring peptide. Methods of altering the DNA sequencesinclude but are not limited to site directed mutagenesis. Examples ofaltered properties include but are not limited to changes in theaffinity of an enzyme for a substrate or a receptor for a ligand.

“Identity” is a measure of the identity of nucleotide sequences or aminoacid sequences. In general, the sequences are aligned so that thehighest order match is obtained. “Identity” per se has an art-recognizedmeaning and can be calculated using published techniques. See, e.g.,:(Computational Molecular Biology, Lesk, A. M., ed. Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds. HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).While there exists a number of methods to measure identity between twopolynucleotide or polypeptide sequences, the term “identity” is wellknown to skilled artisans (Carillo and Lipton, 1988, SIAM J Applied Math48:1073). Methods commonly employed to determine identity or similaritybetween two sequences include, but are not limited to, those disclosedin Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, SanDiego, 1994, and Carillo and Lipton, 1988, SIAM J Applied Math 48:1073.Methods to determine identity and similarity are codified in computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, GCGprogram package (Devereux, et al, 1984, Nucleic Acids Research12(1):387), BLASTN, and FASTA (Altschul, et al., 1990, J Mol. Biol.215:403).

As an illustration, by a polynucleotide having a nucleotide sequencehaving at least, for example, 95% “identity” to a reference nucleotidesequence of SEQ ID NO:1 is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations oralternative nucleotides per each 100 nucleotides of the referencenucleotide sequence of SEQ ID NO:1. In other words, to obtain apolynucleotide having a nucleotide sequence at least 95% identical to areference nucleotide sequence, up to 5% of the nucleotides in thereference sequence may be deleted or substituted with anothernucleotide, or a number of nucleotides up to 5% of the total nucleotidesin the reference sequence may be inserted into the reference sequence.These mutations or alternative nucleotide substitutions of the referencesequence may occur at the 5′ or 3′ terminal positions of the referencenucleotide sequence or anywhere between those terminal positions,interspersed either individually among nucleotides in the referencesequence or in one or more contiguous groups within the referencesequence. One source of such a “mutation” or change which results in aless than 100% identity may occur through RNA editing. The process ofRNA editing results in modification of an mRNA molecule such that use ofthat modified mRNA as a template to generate a cloned cDNA may result inone or more nucleotide changes, which may or may not result in a codonchange. This RNA editing is known to be catalyzed by an RNA editase.Such an RNA editase is RNA adenosine deaminase, which converts anadenosine residue to an inosine residue, which tends to mimic a cytosineresidue. To this end, conversion of an mRNA residue from A to I willresult in A to G transitions in the coding and noncoding regions of acloned cDNA (e.g., see Hanrahan et al, 1999, Annals New York Acad. Sci.868:51–66; for a review see Bass, 1997, TIBS 22: 157–162). Similarly, bya polypeptide having an amino acid sequence having at least, forexample, 95% identity to a reference amino acid sequence of SEQ ID NO:2is intended that the amino acid sequence of the polypeptide is identicalto the reference sequence except that the polypeptide sequence mayinclude up to five amino acid alterations per each 100 amino acids ofthe reference amino acid of SEQ ID NO:2. In other words, to obtain apolypeptide having an amino acid sequence at least 95% identical to areference amino acid sequence, up to 5% of the amino acid residues inthe reference sequence may be deleted or substituted with another aminoacid, or a number of amino acids up to 5% of the total amino acidresidues in the reference sequence may be inserted into the referencesequence. These alterations of the reference sequence may occur at theamino or carboxy terminal positions of the reference amino acid sequenceof anywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence. Again, as noted above,RNA editing may result in a codon change which will result in anexpressed protein which differs in “identity” from other proteinsexpressed from “non-RNA edited” transcripts, which correspond directlyto the open reading frame of the genomic sequence. Therefore, theconcept of nucleic acid sequence identity is applicable to the presentinvention in the context that variations, other than “humanization” ofamino acid residue 74, are within the scope of the present invention solong as those variations do not significantly effect the ability of therespective expressed RAMP1 protein to mimic human RAMP1 when associatedwith a mammalian CRLR protein.

As stated earlier in this section, the present invention also relates torecombinant vectors and recombinant hosts, both prokaryotic andeukaryotic, which contain the substantially purified nucleic acidmolecules disclosed throughout this specification. The nucleic acidmolecules of the present invention encoding a RAMP1 protein, in whole orin part, can be linked with other DNA molecules, i.e, DNA molecules towhich the RAMP1 coding sequence are not naturally linked, to form“recombinant DNA molecules” which encode a respective RAMP1 protein. TheDNA molecules of the present invention can be inserted into vectorswhich comprise nucleic acids encoding RAMP1 or a functional equivalent.These vectors may be comprised of DNA or RNA; for most cloning purposesDNA vectors are preferred. Typical vectors include plasmids, modifiedviruses, bacteriophage, cosmids, yeast artificial chromosomes, and otherforms of episomal or integrated DNA that can encode a RAMP1 protein. Itis well within the purview of the skilled artisan to determine anappropriate vector for a particular gene transfer or other use.Therefore, as with many proteins, it is possible to modify many of theamino acids of RAMP1 protein and still retain substantially the samebiological activity as the wild type protein. Thus this inventionincludes modified RAMP1 polypeptides which have amino acid deletions,additions, or substitutions but that still retain substantially the samebiological activity as a respective, corresponding humanized RAMP1 (i.e,wherein amino acid 74 is a tryptophan residue and any other changes donot significantly effect the ability of the altered RAMP1 to mimic humanpharmacological characteristics as the human CGRP receptor). It isdisclosed herein that the essence of the present invention is theability to humanize a vertebrate RAMP1 protein by altering thevertebrate RAMP1 amino acid sequence at residue 74 to a tryptophanresidue. Therefore, alteration of just a single amino acid resulted in acompletely different, and now predictable, pharmacological profile forsuch a mutated protein. This is a surprising result given thathistorically it was generally accepted that single amino acidsubstitutions do not usually alter the biological activity of a protein(see, e.g., Molecular Biology of the Gene, Watson et al., 1987, FourthEd., The Benjamin/Cummings Publishing Co., Inc., page 226; andCunningham & Wells, 1989, Science 244:1081–1085). To this end, thepresent invention also discloses that minor additional alterations (suchas one, two or several non-silent codon changes) will not effect thehumanizing characteristic of a RAMP1 protein with the Trp74modification, and accordingly, such mutant protein are within the scopeof the present invention. Therefore, the present invention includespolypeptides where one or more additional amino acid substitutions hasbeen made in SEQ ID NOs:2, 4, 6, and/or 8, wherein the polypeptidesstill retain substantially the same biological activity as acorresponding RAMP1 protein. For example, mutation of rat K74W toinclude a mutation at Lys 103 to a Ser residue. This humanized doublemutant shows the same “humanized” pharmacological profile as rat K74W,showing that an additional amino acid substitution does notdeleteriously effect the ability of the K74W to “humanize” the RAMP1protein. The present invention also includes polypeptides where two ormore amino acid substitutions have been made in SEQ ID NOs:2, 4, 6, or8, wherein the polypeptides still retain substantially the samebiological activity as a corresponding RAMP1 protein. In particular, thepresent invention includes embodiments where the above-describedsubstitutions are conservative substitutions. To this end, one ofordinary skill in the art would also recognize that polypeptides thatare functional equivalents of RAMP1 and have changes from the RAMP1amino acid sequence that are small deletions or insertions of aminoacids could also be produced by following the same guidelines, (i.e,minimizing the differences in amino acid sequence between RAMP1 andrelated proteins. Small deletions or insertions are generally in therange of about 1 to 5 amino acids. The effect of such small deletions orinsertions on the biological activity of the modified RAMP1 polypeptidecan easily be assayed by producing the polypeptide synthetically or bymaking the required changes in DNA encoding RAMP1 and then expressingthe DNA recombinantly and assaying the protein produced by suchrecombinant expression. For instance, as long as amino acid residue 74remains in a “humanized” form (i.e., Trp), then minor modifications tothe remainder of the RAMP1 sequence may be generated and are in turneasily tested alongside an expressed CRLR receptor to determine if theexpected human pharmacological profile remains. Furthermore, the presentinvention also includes truncated forms of RAMP1. Such truncatedproteins are useful in various assays described herein, forcrystallization studies, and for structure-activity-relationshipstudies.

The present invention also relates to isolated nucleic acid moleculeswhich are fusion constructions expressing fusion proteins useful inassays to identify compounds which modulate wild-type RAMP1 activity, aswell as generating antibodies against RAMP1. One aspect of this portionof the invention includes, but is not limited to, glutathioneS-transferase (GST)-RAMP1 fusion constructs. Recombinant GST-RAMP1fusion proteins may be expressed in various expression systems,including Spodoptera frugiperda (Sf21) insect cells (Invitrogen) using abaculovirus expression vector (pAcG2T, Pharmingen). Another aspectinvolves RAMP1 fusion constructs linked to various markers, includingbut not limited to GFP (Green fluorescent protein), the MYC epitope,His-tag, and GST. Again, any such fusion constructs may be expressed inthe cell line of interest and used to screen for modulators of one ormore of the RAMP1 proteins disclosed herein, as well as being expressedand purified.

Any of a variety of procedures may be used to clone and generate avertebrate or mammalian RAMP1, such as rat, mouse, human, etc., RAMP1.These methods include, but are not limited to, (1) a RACE PCR cloningtechnique (Frohman, et al., 1988, Proc. Natl. Acad. Sci. USA 85:8998–9002). 5′ and/or 3′ RACE may be performed to generate a full-lengthcDNA sequence. This strategy involves using gene-specificoligonucleotide primers for PCR amplification of RAMP1 cDNA. Thesegene-specific primers are designed through identification of anexpressed sequence tag (EST) nucleotide sequence which has beenidentified by searching any number of publicly available nucleic acidand protein databases; (2) direct functional expression of the RAMP1cDNA following the construction of a RAMP1-containing cDNA library in anappropriate expression vector system; (3) screening a RAMP1-containingcDNA library constructed in a bacteriophage or plasmid shuttle vectorwith a labeled degenerate oligonucleotide probe designed from the aminoacid sequence of the RAMP1 protein; (4) screening a RAMP1-containingcDNA library constructed in a bacteriophage or plasmid shuttle vectorwith a partial cDNA encoding the RAMP1 protein. This partial cDNA isobtained by the specific PCR amplification of RAMP1 DNA fragmentsthrough the design of degenerate oligonucleotide primers from the aminoacid sequence known for other proteins which are related to the RAMP1protein; (5) screening a RAMP1-containing cDNA library constructed in abacteriophage or plasmid shuttle vector with a partial cDNA oroligonucleotide with homology to a RAMP1 protein. This strategy may alsoinvolve using gene-specific oligonucleotide primers for PCRamplification of RAMP1 cDNA identified as an EST as described above; or(6) designing 5′ and 3′ gene specific oligonucleotides using any of thedisclosed mammalian RAMP1 sequences as a template so that either thefull-length cDNA may be generated by known RACE techniques, or a portionof the coding region may be generated by these same known RACEtechniques to generate and isolate a portion of the coding region to useas a probe to screen one of numerous types of cDNA and/or genomiclibraries in order to isolate a full-length version of the nucleotidesequence encoding RAMP1. It is readily apparent to those skilled in theart that other types of libraries, as well as libraries constructed fromother cell types-or species types, may be useful for isolating aRAMP1-encoding DNA or a RAMP1 homologue. Other types of librariesinclude, but are not limited to, cDNA libraries derived from other browndog tick cell types.

It is readily apparent to those skilled in the art that suitable cDNAlibraries may be prepared from cells or cell lines which have RAMP1activity. The selection of cells or cell lines for use in preparing acDNA library to isolate a cDNA encoding RAMP1 may be done by firstmeasuring cell-associated RAMP1 activity using any known assay availablefor such a purpose.

Preparation of cDNA libraries can be performed by standard techniqueswell known in the art. Well known cDNA library construction techniquescan be found for example, in Sambrook et al., 1989, Molecular Cloning: ALaboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. Complementary DNA libraries may also be obtained from numerouscommercial sources, including but not limited to Clontech Laboratories,Inc. and Stratagene.

This invention also includes vectors containing a humanized RAMP1 gene,host cells containing the vectors, and methods of making substantiallypure humanized RAMP1 protein comprising the steps of introducing thehumanized RAMP1 gene into a host cell, and cultivating the host cellunder appropriate conditions such that humanized RAMP1 is produced. Thehumanized RAMP1 so produced may be harvested from the host cells inconventional ways. Therefore, the present invention also relates tomethods of expressing the humanized RAMP1 protein and biologicalequivalents disclosed herein, assays employing these gene products,recombinant host cells which comprise DNA constructs which express theseproteins, and compounds identified through these assays which act asagonists or antagonists of humanized RAMP1 activity.

The cloned humanized RAMP1 cDNA obtained through the methods describedabove may be recombinantly expressed by molecular cloning into anexpression vector (such as pcDNA3.neo, pcDNA3.1, pCR2.1, pBlueBacHis2,pLITMUS28, the pIRES series from Clontech, as well as other examples,listed infra) containing a suitable promoter and other appropriatetranscription regulatory elements, and transferred into prokaryotic oreukaryotic host cells to produce recombinant humanized RAMP1. Expressionvectors are defined herein as DNA sequences that are required for thetranscription of cloned DNA and the translation of their mRNAs in anappropriate host. Such vectors can be used to express eukaryotic DNA ina variety of hosts such as bacteria, blue green algae, plant cells,insect cells and animal cells. Specifically designed vectors allow theshuttling of DNA between hosts such as bacteria-yeast or bacteria-animalcells. An appropriately constructed expression vector should contain: anorigin of replication for autonomous replication in host cells,selectable markers, a limited number of useful restriction enzyme sites,a potential for high copy number, and active promoters. A promoter isdefined as a DNA sequence that directs RNA polymerase to bind to DNA andinitiate RNA synthesis. A strong promoter is one which causes mRNAs tobe initiated at high frequency. To determine the humanized RAMP1 cDNAsequence(s) that yields optimal levels of humanized RAMP1, cDNAmolecules including but not limited to the following can be constructed:a cDNA fragment containing the full-length open reading frame forhumanized RAMP1 as well as various constructs containing portions of thecDNA encoding only specific domains of the protein or rearranged domainsof the protein. All constructs can be designed to contain none, all orportions of the 5′ and/or 3′ untranslated region of a humanized RAMP1cDNA. The expression levels and activity of RAMP1 can be determinedfollowing the introduction, both singly and in combination, of theseconstructs into appropriate host cells. Following determination of thehumanized RAMP1 cDNA cassette yielding optimal expression in transientassays, this humanized RAMP1 cDNA construct is transferred to a varietyof expression vectors (including recombinant viruses), including but notlimited to those for mammalian cells, plant cells, insect cells,oocytes, bacteria, and yeast cells. Techniques for such manipulationscan be found described in Sambrook, et al., supra, are well known andavailable to the artisan of ordinary skill in the art. Therefore,another aspect of the present invention includes host cells that havebeen engineered to contain and/or express DNA sequences encoding thehumanized RAMP1. An expression vector containing DNA encoding ahumanized RAMP1-like protein may be used for expression of humanizedRAMP1 in a recombinant host cell. Such recombinant host cells can becultured under suitable conditions to produce humanized RAMP1 or abiologically equivalent form. Expression vectors may include, but arenot limited to, cloning vectors, modified cloning vectors, specificallydesigned plasmids or viruses. Commercially available mammalianexpression vectors which may be suitable for recombinant humanized RAMP1expression, include but are not limited to, pcDNA3.neo (Invitrogen),pcDNA3.1 (Invitrogen), pCI-neo (Promega), pLITMUS28, pLITMUS29,pLITMUS38 and pLITMUS39 (New England Bioloabs), pcDNAI, pcDNAIamp(Invitrogen), pcDNA3 (Invitrogen), pMClneo (Stratagene), pXT1(Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-1(8-2)(ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199),pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), 1ZD35(ATCC 37565) and the pIRES series (Clontech). Also, a variety ofbacterial expression vectors may be used to express recombinanthumanized RAMP1 in bacterial cells. Commercially available bacterialexpression vectors which may be suitable for recombinant humanized RAMP1expression include, but are not limited to pCR2.1 (Invitrogen), pET11a(Novagen), lambda gt11 (Invitrogen), and pKK223-3 (Pharmacia). Inaddition, a variety of fungal cell expression vectors may be used toexpress recombinant RAMP1 in fungal cells. Commercially available fungalcell expression vectors which may be suitable for recombinant humanizedRAMP1 expression include but are not limited to pYES2 (Invitrogen) andPichia expression vector (Invitrogen). Also, a variety of insect cellexpression vectors may be used to express recombinant protein in insectcells. Commercially available insect cell expression vectors which maybe suitable for recombinant expression of humanized RAMP1 include butare not limited to pBlueBacIII and pBlueBacHis2 (Invitrogen), and pAcG2T(Pharmingen).

Recombinant host cells may be prokaryotic or eukaryotic, including butnot limited to, bacteria such as E. coli, fungal cells such as yeast,mammalian cells including, but not limited to, cell lines of bovine,porcine, monkey and rodent origin; and insect cells. For instance, oneinsect expression system utilizes Spodoptera frugiperda (Sf21) insectcells (Invitrogen) in tandem with a baculovirus expression vector(pAcG2T, Pharmingen). Also, mammalian cells which may be suitable andwhich are commercially available, include but are not limited to, Lcells L-M(TK⁻) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), Saos-2 (ATCCHTB-85), HEK 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61),3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I(ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171) CPAE (ATCCCCL 209), and 293 EBNA cells (Invitrogen).

Expression of humanized RAMP1 DNA may also be performed using in vitroproduced synthetic mRNA. Synthetic mRNA can be efficiently translated invarious cell-free systems, including but not limited to wheat germextracts and reticulocyte extracts, as well as efficiently translated incell based systems, including but not limited to microinjection intofrog oocytes, with microinjection into frog oocytes being preferred.

Following expression of humanized RAMP1 in a host cell, humanized RAMP1protein may be recovered to provide humanized RAMP1 protein in activeform. Several humanized RAMP1 protein purification procedures areavailable and suitable for use. Recombinant humanized RAMP1 protein maybe purified from cell lysates and extracts by various combinations of,or individual application of salt fractionation, ion exchangechromatography, size exclusion chromatography, hydroxylapatiteadsorption chromatography, hydrophobic interaction chromatography, aswell as metal chelate chromotography (e.g., for His-tagged proteins). Inaddition, recombinant humanized RAMP1 protein can be separated fromother cellular proteins by use of an immunoaffinity column made withmonoclonal or polyclonal antibodies specific for full-length humanizedRAMP1 protein, or polypeptide fragments of humanized RAMP1 protein.

Expression of humanized RAMP1 DNA may also be performed using in vitroproduced synthetic mRNA. Synthetic mRNA can be efficiently translated invarious cell-free systems, including but not limited to wheat germextracts and reticulocyte extracts, as well as efficiently translated incell based systems, including but not limited to microinjection intofrog oocytes, with microinjection into frog oocytes being preferred.

Following expression of humanized RAMP1 in a host cell, humanized RAMP1protein may be recovered to provide humanized RAMP1 protein in activeform. Several humanized RAMP1 protein purification procedures areavailable and suitable for use. Recombinant humanized RAMP1 protein maybe purified from cell lysates and extracts by various combinations of,or individual application of salt fractionation, ion exchangechromatography, size exclusion chromatography, hydroxylapatiteadsorption chromatography and hydrophobic interaction chromatography,and metal chelate chromatography. In addition, recombinant humanizedRAMP1 protein can be separated from other cellular proteins by use of animmunoaffinity column made with monoclonal or polyclonal antibodiesspecific for full-length humanized RAMP1 protein, or polypeptidefragments of humanized RAMP1 protein.

The humanized RAMP1 proteins of the present invention may be generatedby techniques known in the art, as shown in Example Sections 1 and 2,for use in an assay procedure with the CRLR GPCR to identify CGRPreceptor modulators (e.g., antagonists of CGRP receptor activity. Ingeneral, an assay procedure to identify such receptor modulators willcontain a humanized CGRP receptor of the present invention, and a testcompound or sample which contains a putative CGRP receptor modulator.The test compounds or samples may be tested directly on, for example,purified receptor protein whether native or recombinant, subcellularfractions of receptor-producing cells whether native or recombinant,and/or whole cells expressing the receptor whether native orrecombinant. The test compound or sample may be added to the receptor inthe presence or absence of a known labeled or unlabelled receptorligand. For instance, recombinant membrane fractions containing ahumanized CRGP receptor can be used to screen for compounds whichinhibit binding of ¹²⁵I-CGRP to the receptor in a radioligand bidingassay. The modulating activity of the test compound or sample may bedetermined by, for example, analyzing the ability of the test compoundor sample to bind to the receptor, activate the receptor, inhibitreceptor activity, inhibit or enhance the binding of other compounds tothe receptor, modify receptor regulation, or modify an intracellularactivity.

The present invention is also directed to methods for screening forcompounds which modulate the expression of DNA or RNA encoding ahumanized CGRP receptor as well as the function of a humanized CGRPreceptor in vivo. Compounds which modulate these activities may be DNA,RNA, peptides, proteins, or non-proteinaceous organic molecules.Compounds may modulate by increasing or attenuating the expression ofDNA or RNA encoding CRLR and/or humanized RAMP1 receptor respectively,or the function either protein. Compounds that modulate the expressionof DNA or RNA encoding the CGRP receptor or the function of thisreceptor may be detected by a variety of assays. The assay may be asimple “yes/no” assay to determine whether there is a change inexpression or function. The assay may be made quantitative by comparingthe expression or function of a test sample with the levels ofexpression or function in a standard sample.

The following examples are presented by the way of illustration and,because various other embodiments will be apparent to those in the art,the following is not to be construed as a limitation on the scope of theinvention.

EXAMPLE 1 Characterization of Various Mammalian and Humanized RAMP1cDNAs

Marmoset and Cynomolgous RAMP1 cDNA Cloning—A partial marmoset RAMP1cDNA and cynomolgous cDNA were isolated from frontal brain cDNA usingthe polymerase chain reaction (PCR). The PCR primers were based uponhuman RAMP1 (5′-CTGCCAGGAGGCTAACTACG-3′ [SEQ ID NO:25] and5′-CACGATGAAGGGGTAGAGGA-3′ [SEQ ID NO:26]). Amplification reactionsconsisted of 40 cycles of 45 sec at 94° C., 45 sec at 58° C., and 1 minat 72° C. and were carried out according to the manufacturer'srecommended protocol for PLATINUM Taq PCR DNA polymerase (Invitrogen).Multiple subclones were sequenced to rule out potential errors.

Expression Constructs, Chimeras, and Mutagenesis—Human and rat cDNAs forCRLR were subcloned as 5′NheI and 3′NotI fragments into pcDNA3.1/Zeo⁽⁺⁾(Invitrogen). Human RAMP1 (hRAMP1) was provided in the expression vectorpcDNA3.1⁽⁺⁾ (Invitrogen). Rat RAMP1 (rRAMP1) cloning was as disclosed inOliver et al., 2001, Eur. J. Neuroscience 14: 618–628, herebyincorporated by reference. The cDNA was subcloned as a 5′NotI and3′BamHI fragment into pcDNA3.1/Hygro(−) (Invitrogen). Table 3 showsvarious wild type mammalian RAMP1 nucleotide and amino acid sequences.FIG. 4 also shows an alignment of amino acid sequences through the“humanizing residue” at residue #74, including human and marmoset (Trp),rat and mouse (Lys, which may be mutagenized to Trp) and pig (Arg, whichmay be mutaginized to Trp). FIG. 1 shows the alignment of the fulllength amino acid sequences for human, rat and mouse RAMP1.

TABLE 3 Human RAMP1 Nucleotide Sequence ATGGCCCGGG CCCTGTGCCG CCTCCCGCGGCGCGGCCTCT GGCTGCTCCT GGCCCATCAC (SEQ ID NO:15) CTCTTCATGA CCACTGCCTGCCAGGAGGCT AACTACGGTG CCCTCCTCCG GGAGCTCTGC CTCACCCAGT TCCAGGTAGACATGGAGGCC GTCGGGGAGA CGCTGTGGTG TGACTGGGGC AGGACCATCA GGAGCTACAGGGAGCTGGCC GACTGCACCT GGCACATGGC GGAGAAGCTG GGCTGCTTCT GGCCCAATGCAGAGGTGGAC AGGTTCTTCC TGGCAGTGCA TGGCCGCTAC TTCAGGAGCT GCCCCATCTCAGGCAGGGCC GTGCGGGACC CGCCCGGCAG CATCCTCTAC CCCTTCATCG TGGTCCCCATCACGGTGACC CTGCTGGTGA CGGCACTGGT GGTCTGGCAG AGCAAGCGCA CTGAGGGCATTGTGTAG Human RAMP1 Amino Acid Sequence MARALCRLPR RGLWLLLAHH LFMTTACQEANYGALLRELC LTQFQVDMEA VGETLWCDWG (SEQ ID NO:16) RTIRSYRELA DCTWHMAEKLGCFWPNAEVD RFFLAVHGRY FRSCPISGRA VRDPPGSILY PFIVVPITVT LLVTALVVWQSKRTEGIV Rat RAMP1 Nucleotide Sequence ATGGCCCCCG GCCTGCGGGG CCTCCCGCGGCGCGGCCTCT GGCTGCTGCT GGCTCATCAT (SEQ ID NO:17) CTCTTCATGG TCACTGCCTGCCGGGACCCT GACTATGGTA CTCTCATCCA GGAGCTGTGT CTCAGCCGCT TCAAAGAGGACATGGAGACC ATAGGGAAGA CTCTGTGGTG TGACTGGGGA AAGACCATAG GGAGCTATGGGGAGCTCACT CACTGCACCA AACTCGTGGC AAACAAGATT GGCTGTTTCT GGCCCAATCCGGAAGTGGAC AAGTTCTTCA TTGCTGTCCA CCACCGCTAC TTCAGCAAGT GCCCAGTCTCGGGCAGGGCC CTGCGGGACC CTCCCAACAG CATCCTCTGC CCTTTCATTG TGCTCCCCATTACGGTCACA CTGCTCATGA CTGCCCTGGT GGTCTGGAGG AGCAAGCGCA CAGAGGGCATCGTGTAG Rat RAMP1 Amino Acid Sequence MAPGLRGLPR RGLWLLLAHH LFMVTACRDPDYGTLIQELC LSRFKEDMET IGKTLWCDWG (SEQ ID NO:18) KTIGSYGELT HCTKLVANKIGCFWPNPEVD KFFIAVHHRY FSKCPVSGRA LRDPPNSILC PFIVLPITVT LLMTALVVWRSKRTEGIV Mouse RAMP1 Nucleotide Sequence ATGGCCCCGG GCCTGCGGGGCCTCCCGCGG TGCGGCCTCT GGCTGCTGCT GGCTCACCAT (SEQ ID NO:19) CTCTTCATGGTCACTGCCTG CCGGGACCCT GACTATGGGA CTCTCATCCA GGAGCTGTGC CTCAGCCGCTTCAAGGAGAA CATGGAGACT ATTGGGAAGA CGCTATGGTG TGACTGGGGA AAGACCATACAGAGCTATGG GGAGCTCACT TACTGCACCA AGCACGTGGC GCACACGATT GGCTGTTTCTGGCCCAATCC GGAAGTGGAC AGATTCTTCA TCGCTGTCCA CCATCGATAC TTCAGCAAGTGCCCCATCTC GGGCAGGGCC CTGCGGGACC CTCCCAACAG CATCCTCTGC CCTTTCATTGCGCTCCCCAT TACGGTCACG CTGCTCATGA CTGCACTGGT GGTCTGGAGG AGCAAGCGCACAGAGGGCAT CGTGTAG Mouse RAMP1 Amino Acid Sequence MAPGLRGLPR CGLWLLLAHHLFMVTACRDP DYGTLIQELC LSRFKENMET IGKTLWCDWG (SEQ ID NO:20) KTIQSYGELTYCTKHVAHTI GCFWPNPEVD RFFIAVHHRY FSKCPISGRA LRDPPNSILC PFIALPITVTLLMTALVVWR SKRTEGIV Cynomolgous RAMP1 Nucleotide Sequence (Partial)GTGCCCTCCT CCAGGAGCTC TGCCTCACCC AGTTCCAGGT AGACATGGAG GCCGTCGGGG (SEQID NO:21) AGACGCTGTG GTGTGACTGG GGCAGGACCA TCGGGAGCTA CAGGGAGCTGGCCGACTGCA CCTGTCACAT GGCGGAGAAG CTAGGCTGCT TCTGGCCCAA CGCAGAGGTGGACAGGTTCT TCCTGGCAGT GCACGGGCAC TACTTCAGGG CCTGCCCCAT CTCAGGCAGGGCCGTGCGGG ACCCGCCTGG CAGCG Cynomolgous RAMP1 Amino Acid Sequence(Partial) ALLQELCLTQ FQVDMEAVGE TLWCDWGRTI GSYRELADCT CHMAEKLGCFWPNAEVDRFF (SEQ ID NO:22) LAVHGHYFRA CPISGRAVRD PPGS Porcine (Pig) RAMP1Nucleotide Sequence (Partial) AGGACCATCA GGAGCTATAA AGACCTCTCAGACTGCACCA GGCTCGTGGC GCAAAGGCTG (SEQ ID NO:23) GACTGCTTCT GGCCCAACGCGGCGGTGGAC AAGTTCTTCC TGGGAGTCCA CCAGCAGTAC TTCAGAAACT GCCCCGTCTCCGGCAGGGCC TTGCAGGACC CGCCCAGCAG CGTCCTCTGC CCCTTCATCG TCGTCCCCATCCTGGCGACC CTGCTCATGA CCGCACTGGT GGTCTGGCAG Porcine (Pig) RAMP1 AminoAcid Sequence (Partial) RTIRSYKDLS DCTRLVAQRL DCFWPNAAVD KFFLGVHQQYFRNCPVSGRA LQDPPSSVLC (SEQ ID NO:24) PFIVVPILAT LLMTALVVWQ

Two human/rat chimeric RAMP1 cDNAs were constructed by using restrictionfragments of the corresponding native cDNAs. Chimera 1 was created byreplacing the nucleotides coding for the first 66 amino acids of rRAMP1with the corresponding nucleotides of hRAMP1 by using the BsgIrestriction site along with a NheI site located in the cloning vector.Chimera 2 was created by replacing the nucleotides coding for the first112 amino acids of rRAMP1 with the corresponding nucleotides of hRAMP1by using the SanDI restriction site along with a NheI site located inthe cloning vector.

Rat RAMP1 site-directed mutagenesis was performed by using the QuickChange Site-directed Mutagenesis Kit (Stratagene) according to themanufacturer's instructions. Lysine at position 74 of rRAMP1 wasreplaced with the corresponding human/marmoset amino acid tryptophanutilizing two complementary mutant oligonucleotide primers(5′-CCACTCACTGCACCTGGCTCGTGGCAAACAAG-3′ [SEQ ID NO:27] and5′-CTTGTTTGCCACGAGCCAGGTGCAGTGAGTGAG-3′ [SEQ ID NO:28]) and the rRAMP1expression vector construct as template. This mutation was accomplishedby substituting the codon TGG corresponding to tryptophan (rK74W RAMP1).All constructs were sequenced bidirectionally with 100% coverage in eachdirection.

Cell Culture and DNA Transfection—293 EBNA cells were cultured in DMEMwith 4.5 g/L glucose, 1 mM sodium pyruvate and 2 mM glutaminesupplemented with 10% Fetal Bovine Serum (FBS), 100 units/mL penicillinand 100 μg/mL streptomycin, and maintained at 37° C. and 95% humidity.Cells were subcultured by treatment with 0.25% trypsin with 0.1% EDTA inHBSS.

Twenty-four hours prior to transfection, the cells were seeded at2.0×10⁷/dish in 500 cm² dishes. The following day, the cells were re-fedwith fresh growth medium 1 hour before transfection. Transfections wereperformed by combining 60 μg/dish DNA with 180 μg/dish Lipofectamine2000 (Life Technologies). cDNA's for CRLR and RAMP1 in the mammalianexpression vector pcDNA3.1 were co-transfected in equal amounts. Thetransfection cocktail was added directly to the medium and this mixturewas replaced with fresh medium 24 hours later. The cells were harvestedfor membranes 48 hours post-transfection.

Membrane Preparation and Radioligand Binding Studies—Transientlytransfected 293 EBNA cells were washed once with PBS and harvested inharvest buffer containing 50 mM HEPES, 1 mM EDTA and Complete proteaseinhibitors (Roche). The cell suspension was disrupted with a laboratoryhomogenizer and centrifuged at 48000 g to isolate membranes. The pelletswere re-suspended in harvest buffer plus 250 mM sucrose. Membranes werestored at −70° C. as aliquots.

For binding assays, 1.5–25 μg of membranes (dependent upon receptorexpression levels) were incubated for 3 hours at room temperature inbinding buffer (10 mM HEPES, 5 mM MgCl₂, 0.2% BSA) containing 10 pM¹²⁵I-hCGRP (Amersham) in a total volume of 1 mL. Similar results wereobtained by using ¹²⁵I-rCGRP (Amersham). Incubations were terminated byfiltration through GF/B 96-well filter plates that had been blocked with0.5% polyethyleneimine. Non-specific binding was determined by using afinal concentration of 100 nM hCGRP (for peptide assays) or 300 nMBIBN4096BS (for small molecule assays). Data were analyzed by usingGraphPad Prism. Dose response curves were plotted and IC₅₀ valuesdetermined from a 4-parameter fit as defined by the equationy=((a−d)/(1+(x/c)^(b))+d, where y=response, x=dose, a=max response,d=min response, c=inflection point and b=slope. Data reported in Table4–6 are from a single experiment, but are representative of 2–3replicates.

Western Blotting—Membranes expressing rCRLR were treated separately witheither Endoglycosidase F1 or Peptide-N-Glycosidase F (Calbiochem)overnight at 37° C. After the addition of protein gel loading buffer,the samples were heated at 70° C. for 10 min, then loaded onto a 4–12%gradient NuPAGE Bis-Tris polyacrylamide gel (Invitrogen). Followingelectrophoresis, the separated proteins were transferred to a 0.45 μmnitrocellulose membrane. Rat CRLR was detected by using theWesternBreeze Immunodetection kit (Invitrogen) with affinity purifiedrabbit anti-rat CRLR (Alpha Diagnostic International).

Results—Small molecule antagonists of the CGRP receptor have been shownto exhibit species selective pharmacology (Doods, et al., 2000, Br. J.Pharmacol. 129, 420–423; Edvinsson, et al., 2001, Eur. J. Pharmacol.415: 39–44; Hasbak, et al., 2001, Br. J. Pharmacol. 133: 1405–1413).Protein sequence alignment reveals that while human and rat CRLR are 91%homologous, human and rat RAMP1 share only 71% homology. BIBN4096BS wasreported to exhibit 200-fold higher affinity for the human CGRP receptorthan for the rat receptor (Doods, et al., id.). This observationsuggested that the pharmacological differences could be a result of thesequence dissimilarity of either protein, or may result from a combinedeffect of differences in both CRLR and RAMP1 sequences. In order tofirst determine if the species selectivity is derived from either CRLRitself, or its accessory protein RAMP1, hybrid human/rat CGRP receptorswere created by transiently transfecting cDNA's coding for human CRLRwith rat RAMP1 and vice versa in 293 EBNA cells. The cells wereharvested and cell membranes were prepared for subsequent competitiveligand binding experiments. As expected, the small molecule antagonistsCompound 1 and BIBN4096BS had lower affinity for rCRLR/rRAMP1 than forthe transfected human CGRP receptor (Table 4; see FIG. 2 for structureof BIBN4096BS and Compounds 1 and 2). However, the peptide antagonistCGRP₈₋₃₇ displayed similar affinities for CGRP receptors from human andrat, with IC₅₀ values of 2.8 and 2.0 nM, respectively. In 293 EBNAmembranes expressing rCRLR/rRAMP1, ²⁵I-hCGRP binding was inhibited byCompound 1 and BIBN4096BS with IC₅₀ values of 15,000 and 6.9 nMrespectively. In contrast, recombinantly expressed rCRLR/hRAMP1 showedhuman-like pharmacology toward Compound 1 and BIBN4096BS with IC₅₀'s of190 and 0.41 nM respectively (Table 4). Likewise, the IC₅₀ values forCompound 1 and BIBN4096BS for hCRLR/rRAMP1 were similar to thoseobserved for the native rat receptor. These results demonstrated thatRAMP1 determines the affinity of BIBN4096BS and Compound 1 for human andrat CGRP receptors. The species origin of CRLR in these hybrid receptorshad little or no effect on the small molecule antagonist affinities.

TABLE 4 Summary of competitive binding experiments on membranesexpressing mixed species CRLR/RAMP1 receptor complexes. hCRLR and rCRLRwere transiently transfected into 293 EBNA cells along with either humanor rat RAMP1. Membranes were prepared 48 hours post-transfection. IC₅₀,nM Compound 1 BIBN4096BS hCRLR/hRAMP1 150 0.16 rCRLR/rRAMP1 15,000 6.9rCRLR/hRAMP1 190 0.41 hCRLR/rRAMP1 24,000 6.1RAMPs are accessory proteins predicted to contain a large extracellularN-terminal domain and a single transmembrane (TM) spanning domain. Toelucidate the region of RAMP1 that is directly involved in determiningthe affinities of BIBN4096BS and Compound 1, human/rat RAMP1 chimeraswere generated. Chimera 1 was created by replacing the first 66 aminoacids of rRAMP1 with the corresponding hRAMP1 sequence (FIG. 3).Conversely, replacement of the first 112 amino acids of rRAMP1 with thehuman sequence produced Chimera 2. These constructs were then used fortransient transfections in similar experiments as described above. Inmembranes expressing rCRLR with Chimera 1, ¹²⁵I-hCGRP binding wasinhibited by Compound 1 and BIBN4096BS with IC₅₀ values of 9,000 and 4.8nM respectively (Table 5). These results were similar to those obtainedfor rCRLR/rRAMP1. By contrast, when rCRLR was co-expressed with Chimera2, the resulting IC₅₀'s were similar to those obtained for hCRLR/hRAMP1.These studies demonstrated that amino acids 66–112 in the extracellulardomain of RAMP1 were responsible for modulating the affinity ofBIBN4096BS and Compound 1 for CRLR/RAMP1.

TABLE 5 Summary of competitive binding experiments on membranesexpressing rCRLR with the RAMP1 Chimeras. IC₅₀, nM Compound 1 BIBN4096BShCRLR/hRAMP1 150 0.16 rCRLR/rRAMP1 15,000 6.9 rCRLR/Chimera 1 9,000 4.8rCRLR/Chimera 2 150 0.16

The identification of amino acids 66–112 of RAMP1 as the critical regiondetermining CGRP receptor pharmacology allows for the possibility thatthe species selectivity might be governed by specific amino acidresidues. A partial marmoset RAMP1 cDNA was cloned and the sequencecompared with that from human and the other available RAMP1 sequences.Protein sequence alignment revealed fourteen residues that wereidentical in human and marmoset but different from that found in rat,mouse and pig (FIG. 4). Amino acid 74 was targeted as a potentialmutagenesis target, since the human and marmoset sequences containedtryptophan at this position, but a basic residue was found in the threeother species. Subsequently, lysine at position 74 of rRAMP1 wasreplaced with the corresponding human/marmoset amino acid tryptophan.This construct was then co-transfected with rCRLR in 293 EBNA cells.Competitive binding experiments demonstrated that human-like receptorpharmacology could be rescued by co-expression of rCRLR with rK74W RAMP1(Table 6). The IC₅₀ of BIBN4096BS for rCRLR/rK74W RAMP1 was similar tothat observed for hCRLR/hRAMP1, 0.08 versus 0.02 nM, respectively, andwas significantly more potent than the affinity for the native ratreceptor, 5.5 nM. A similar trend was observed for Compound 2. Compound1 exhibited >10-fold higher affinity for the rCRLR/rK74W RAMP1 receptorthan for the native human receptor, perhaps due to favorableinteractions between the dibromotyrosyl moiety and the tryptophan in theRAMP1 mutant. These results suggested that the affinities of these smallmolecule antagonists for the CGRP receptor were heavily influenced bythe nature of amino acid 74 of RAMP1.

TABLE 6 Summary of competitive binding experiments on membranesexpressing rCRLR and mutant rK74W RAMP1. IC₅₀, nM Compound 1 Compound 2BIBN4096BS hCRLR/hRAMP1 270 104 0.02 rCRLR/rRAMP1 20,000 >20,000 5.5rCRLR/rK74W 19 120 0.08 RAMP1

One of the demonstrated functions of RAMPs is to ensure proper cellsurface targeting of CRLR. The functional significance of glycosylationwas therefore addressed because the glycosylation state of the rat CGRPreceptor had not been characterized previously; furthermore, thepossibility existed that rat and human RAMP1 resulted in differentialglycosylation of CRLR and that this effect determined the observeddifferences in antagonist affinities. Using an antibody to rCRLR anddeglycosylation enzymes, the glycosylation state of rCRLR associatedwith rat or human RAMP1 was determined. The membranes from thecompetitive binding experiments (rCRLR/rRAMP1, rCRLR/hRAMP1 and controlrCRLR/pcDNA3.1) were treated with Peptide-N-Glycosidase F (PNGase F) andEndoglycosidase F1 (Endo F1). PNGase F catalyzes the hydrolysis ofmature glycoproteins, whereas Endo F1 cleaves N-linked high mannose andhybrid oligosaccharides, but not complex oligosaccharides. Thus, themolecular weight of a glycosylated receptor will decrease aftertreatment with PNGase F and a receptor with complex glycosylation willresist Endo F1 cleavage. Co-expression of rCRLR with either human or ratRAMP1 produced M_(r) species of 55 and 68 kDa, which were reduced to asingle 42 kDa species following PNGase F treatment (FIG. 5).Furthermore, the 68 kDa species represented a mature glycoprotein, asdemonstrated by its resistance to Endo F1 cleavage. The negative controlrCRLR alone resulted in background levels of the 55 kDa species,possibly resulting from interaction of transfected CRLR with low levelsof endogenous RAMP1. The 55 kDa species likely represents a coreglycosylated form of the receptor. These results indicated that theco-expression of either human or rat RAMP1 with rat CRLR resulted insimilar levels of complex glycosylation.

EXAMPLE 2

A mouse cDNA for CRLR was isolated from mouse brain cDNA using thepolymerase chain reaction (PCR). PCR primers(5′-TAGCTAGCGCCACCATGGATAAAAAGCATATAC [SEQ ID NO:29] and5′-CGGGATCCTGGCTATCCAATCTTTTGGC-3′ [SEQ ID NO:30]) were based uponGenbank accession number AF209905. Engineered 5′NheI and 3′BamHI siteswere utilized for subcloning into the expression vectorpcDNA3.1/Hygro(−) (Invitrogen). A mouse cDNA for RAMP1 was isolated frommouse brain cDNA utilizing PCR. PCR primers(5′-ATGCGGCCGCGTGGGGCTCTGCTTGCCATG-3′ [SEQ ID NO:31] and5′-CGGGATCCCTCATCACCTGGGATACCTAC-3′ [SEQ ID NO:32]) were based upon thepublished mouse RAMP1 sequence (Knut, et al., 2000, Mol. Cell.Endocrinol. 162: 35–43). Engineered 5′NotI and 3′BamHI sites wereutilized for subcloning into the expression vector pcDNA3.1/Hygro(−)(Invitrogen). Mouse RAMP1 site-directed mutagenesis was performed by thesame method employed in EXAMPLE 1. The mouse RAMP1 expression vectorconstruct was used as template utilizing two complementary mutantoligonucleotide primers (5′-GCTCACTTACTGCACCTGGCACGTGGCGCACACG [SEQ IDNO:33] and 5′-CGTGTGCGCCACGTGCCAGGTGCAGTAAGTGAGC [SEQ ID NO:34]). Thismutation was accomplished by substituting a TG at positions 1 and 2 ofthe mouse lysine codon (AAG) resulting in the tryptophan codon TGG(mK74W RAMP1). Cell culture, DNA transfection, membrane preparation, andradioligand biding studies were carried out as in EXAMPLE 1.

Competitive binding experiments demonstrated that human-like receptorpharmacology could be rescued by co-expression of mCRLR with mK74W RAMP1(Table 7). The IC₅₀ of BIBN4096BS for mCRLR/mK74W RAMP1 was similar tothat observed for hCRLR/hRAMP1, 0.1 versus 0.02 nM, respectively, andwas significantly more potent than the affinity for the native mousereceptor, 8.5 nM. A similar trend was observed for Compound 2. Compound1 exhibited >10-fold higher affinity for the mCRLR/mK74W RAMP1 receptorthan for the native human receptor as was also observed with therCRLR/rK74W RAMP1 receptor.

TABLE 7 Summary of competitive binding experiments on membranesexpressing mCRLR and mutant mK74W RAMP1. IC₅₀, nM Compound 1 Compound 2BIBN4096BS hCRLR/hRAMP1 150 70 0.02 mCRLR/mRAMP1 >20,000 >20,000 8.5mCRLR/mK74W 13 310 0.1 RAMP1

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. A purified nucleic acid molecule encoding a mammalianreceptor-activity modifying protein 1 which comprises a nucleotidesequence selected from the group consisting of SEQ ID NO: 1 and
 3. 2. Anexpression vector for expressing a mammalian receptor-activity modifyingprotein 1 in a recombinant isolated host cell wherein said expressionvector comprises a nucleic acid molecule of claim
 1. 3. A isolated hostcell which expresses a recombinant mammalian receptor-activity modifyingprotein 1 wherein said host cell contains the expression vector of claim2.
 4. A process for expressing a mammalian receptor-activity modifyingprotein 1 in a recombinant isolated host cell, comprising: (a)transfecting the expression vector of claim 2 into a suitable host cell;and, (b) culturing the host cells of step (a) under conditions whichallow expression of said protein from said expression vector.
 5. Apurified mammalian receptor-activity modifying protein 1 comprising anamino acid sequence selected from the group consisting of SEQ ID NOs: 2and
 4. 6. A membrane preparation comprising the mammalianreceptor-activity modifying protein 1 purified from the recombinant hostcell of claim
 3. 7. The membrane preparation of claim 6 which furthercomprises a mammalian calcitonin-receptor-like receptor protein, suchthat a receptor-activity modifying protein 1-calcitonin-receptor-likereceptor protein complex, in the presence of Compound 1, Compound 2, andBIBN4096BS, exhibits IC₅₀ values comparable to that of the humancomplex.
 8. The membrane preparation of claim 7 wherein thecalcitonin-receptor-like receptor protein is selected from the groupconsisting of SEQ ID NOs: 10, 12 and 14.